- Miscellaneous 594
- Geoduck Genome Sequencing 68
- Olympia oyster reciprocal transplant 67
- Olympia Oyster Genome Sequencing 62
- PROPS 36
- Computer Servicing 24
- Crassostrea gigas larvae OA (2011) bisulfite sequencing 24
- Samples Submitted 23
- LSU C.virginica Oil Spill MBD BS Sequencing 22
- 2bRAD Library Tests for Sequencing at Genewiz 22
- Genotype-by-sequencing at BGI 22
- Goals 20
- Protein expression profiles during sexual maturation in Geoduck 14
- Samples Received 13
- Lineage-specific DNA methylation patterns in developing oysters 11
- BS-seq Libraries for Sequencing at Genewiz 11
- Sea star RNA-seq 10
- MBD Enrichment for Sequencing at ZymoResearch 8
- Reagent Prep 7
- SRA Submissions 5
- Myostatin Interacting Proteins 3
- Tanner Crab RNAseq 3
- 1
- 1
- Olypia Oyster Genome Sequencing 1
Miscellaneous
Sequence Subsetting - Pgenerosa_v70 Genome Assembly with faidx
Steven asked to subset geoduck assembly in to >10kbp and >30kbp groups. Here are the commands, using faidx:
DNA Isolation and Quantification - Ronit’s C.gigas Ploidy Ctenidia
Last week, I isolated DNA from all of Ronti’s ctenidia samples, however one sample (D18-C) didn’t isolate properly. So, I performed another isolation procedure with another section of frozen tissue. Tissue was excised from frozen tissue block via razor blade (weight not recorded) and pulverized under liquid nitrogen. Samples were incubated O/N @ 37oC (heating block) in 350uL of MB1 Buffer + 25uL Proteinase K, per the E.Z.N.A. Mollusc DNA Kit (Omega) instructions.
DNA Isolation - C.gigas Ploidy Experiment Ctenidia
Isolated DNA from the remaining ctenidia tissue samples from Ronit’s experiment (Google Sheet).
Gene Prediction - HiSeqX Metagenomics from Geoduck Water Using MetaGeneMark on Mox
After assembline the metagenomic data yesterday, I needed to predict some genes. I did this using MetaGeneMark (v.3.38) and ran it on Mox.
Metagenome Assembly - P.generosa Water Sample Trimmed HiSeqX Data Using Megahit on Mox
Attempting an assembly of our geoduck metagenomics HiSeqX data using Megahit (v.1.1.4) on a Mox node.
VCF Splitting - C.virginica VCF Using BCFtools
DNA Isolation - C.gigas Ploidy Experiment Ctenidia
Yesterday, Ronit initiated DNA isolation from ctenidia samples from his experiment (Google Sheet) from the following four samples:
qPCR - Relative mitochondrial abundance in C.gigas diploids and triploids subjected to acute heat stress via COX1
Using the C.gigas cytochrome c oxidase (COX1) primers (SR IDs: 1713, 1714)I designed the other day, I ran a qPCR on a subset of Ronit’s diploid/triploid control/heat shocked oyster DNA that Shelly had previously isolated and performed global DNA methylation assay. The goal is to get a rough assessment of whether or not there appear to be differences in relative mitochondrial abundances between these samples.
BLASTx - Clupea pallasii (Pacific herring) liver and testes transcriptomes on Mox
Apparently we’ve had some interest in the Pacific herring transcriptomes (liver and testes) we produced many years ago. As such, Steven asked that I do a quick BLASTx to help annotate these two transcriptomes.
FastQC and Trimming - Metagenomics (Geoduck) HiSeqX Reads from 20180809
Steven tasked me with assembling our geoduck metagenomics HiSeqX data. The first part of the process is examining the quality of the sequencing reads, performing quality trimming, and then checking the quality of the trimmed reads. It’s also possible (likely) that I’ll need to run another round of trimming. The process is documented in the Jupyter Notebook linked below. After these reads are cleaned up, I’ll transfer them over to our HPC nodes (Mox) and try assembling them.
Primer Design - Gigas COX1 using Primer3
We’re attempting a quick & dirty comparison of relative mitochondria counts between diploid and triploid C.gigas, so needed a single-copy mitochondrial gene target for qPCR. Selected cytochrome c oxidase subunit 1 (COX1), based on a quick glance at the NCBI mt genome browser for C.gigas NC_001276.
qPCR - Geoduck gonad cDNA with vitellogenin primers
Earlier today I made some cDNA from geoduck gonad RNA for use in this qPCR to test out the vitellogenin primers I designed on 20181129
Reverse Transcription - Geoduck gonad RNA pool
To be able to actually test the vitellegenin primers I made, I needed some geoduck cDNA.
Primer Design - Geoduck Vitellogenin using Primer3
In preparation for designing primers for developing a geoduck vitellogenin qPCR assay, I annotated a de novo geoduck transcriptome assembly last week. Next up, identify vitellogenin genes, design primers, confirm their specificity, and order them!
Annotation - Geoduck Transcritpome with TransDecoder
I was tasked with generating some qPCR primers to analyze vitellogenin expression in geoduck. In order to do so, I needed to annotate a geoduck transcriptome in order to identify potential vitellogenin genes. I had previously assembled a geoduck transcriptome. For annotation, I used TransDecoder (v5.5.0). The annotation was run on our Mox HPC node.
Reverse Transcription - Ronit’s C.gigas DNased RNA from 20181115
Quantified Ronit’s DNased RNA earlier today and proceeded with reverse transcription using 100ng of DNased RNA with oligo dT primers (Promega) and M-MLV reverse transcriptase (Promega) according to the manufacturer’s protocol.
RNA Quantification - Ronit’s C.gigas DNased RNA from 20181115
After confirming Ronit’s DNased RNA was free of gDNA, I quantified the DNased RNA from 20181115 using the Roberts Lab Qubit 3.0 and the Qubit hsRNA Assay.
qPCR - Ronit’s DNased C.gigas RNA with Elongation Factor Primers
After DNasing Ronit’s RNA earlier today, I needed to verify the RNA was free of any contaminating gDNA.
DNase - Ronit’s C.gigas Ctenidia RNA
DNA Isolation and Quantification - Lotterhos C.virginica Mantle DNA
Isolated DNA from the Lotterhos Crassostrea virginica mantle samples received on 20181017 using the E.Z.N.A. Mollusc Kit, with the following modifications/notes:
Computing - Install NCBI nr nt BLAST Database on Mox
Per this issue on GitHub, I installed the pre-formatted NCBI non-redudant (nr) nucleotide (nt) database on Mox.
qPCRs - Ronit’s C.gigas ploidy/dessication/heat stress cDNA (1:5 dilution)
IMPORTANT: The cDNA used for the qPCRs described below was a 1:5 dilution of Ronit’s cDNA made 20181017 with the following primers! Diluted cDNA was stored in his -20oC box with his original cDNA.
Reverse Transcription - Ronit’s C.gigas DNased ctenidia RNA
Proceeded with reverse transcription of [Ronit’s DNased ctenidia RNA (from 20181016)(2018-10-16-dnase-treatment-ronits-c-gigas-ploiyddessication-ctenidia-rna.html).
qPCR - Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with elongation factor primers
After I figured out the appropriate DNA and primers to use to detect gDNA in Crassostrea gigas samples, I checked Ronit’s [DNased ctenidia RNA (from 20181016)(2018-10-16-dnase-treatment-ronits-c-gigas-ploiyddessication-ctenidia-rna.html) for residual gDNA.
qPCR – C.gigas primer and gDNA tests with 18s and EF1 primers
The qPCR I ran earlier today to check for residual gDNA in Ronit’s DNased RNA turned out terribly, due to a combination of bad primers and, possibly, bad gDNA.
qPCR - Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with 18s primers
After DNasing Ronit’s RNA earlier today, I needed to check for any residual gDNA.
DNase Treatment - Ronit’s C.gigas Ploiyd/Dessication Ctenidia RNA
After quantifying Ronit’s RNA earlier today, I DNased them using the Turbo DNA-free Kit (Ambion), according to the manufacturer’s standard protocol.
RNA Quantification - Ronit’s C.gigas Ploidy/Dessication RNA
Last Friday, Ronit quantified 1:10 dilutions of the RNA I isolated on 20181003 and the RNA he finished isolating on 20181011, but two of the samples (D11-C, T10-C) were still too concentrated.
Data Received - Chionoecetes bairdi RNAseq & FastQC Analysis
We received Grace’s 100bp PE NovaSeq (Illumian) RNAseq data from the Northwest Genomics Center today.
VCF Splitting with bcftools
RNA Isolation - Tanner Crab Hemolymph Pellet in RNAlater using TriReagent
I previously isolated RNA from crab hemolymp from a lyophilized sample using TriReagent and Grace recently tried isolating RNA from crab hemolyph pellet (non-lyophilized) using TriReagent. The results for her extractions weren’t so great, so I’m giving it a shot with the following samples:
RNA Isolation - Ronit’s C.gigas diploid/triploid dessication/heat shock ctenidia tissues
Isolated RNA from a subset of Ronit’s Crassostrea gigas ctenidia samples (see Ronit’s notebook for experiment deets):
Installation - Microsoft Machine Learning Server (Microsoft R Open) on Emu/Roadrunner R Studio Server
Steven recently saw an announcement that Microsoft R Open now handles multi-threaded processing (default R does not), so we were interested in trying it out. I installed MLR/MRO on Emu/Roadrunner (Apple Xserve; Ubuntu 16.04). Followed the Microsoft installation directions for Ubuntu. In retrospect, I think I could’ve just installed MRO, but this gets the job done as well and won’t hurt anything.
Transcriptome Alignment & Bedgraph – Olympia oyster transcriptome with Olurida_v080 genome assembly
Bedgraph – Olympia oyster transcriptome with Olurida_v081 genome assembly
I took the sorted BAM file from yesterday’s corrected RNAseq genome alignment and converted it to a bedgraph using BEDTools genomeCoverageBed tool.
Transcriptome Alignment – Olympia oyster RNAseq reads aligned to genome with HISAT2
Yesterday’s attempt at producing a bedgraph was a failure and a prodcuct of a major brain fart. The worst part is that I was questioning what I was doing the entire time, but still went through with the process! Yeesh!
Bedgraph - Olympia oyster transcriptome (FAIL)
Progress on generating bedgraphs from our Olympia oyster transcriptome continues.
Transcriptome Alignment - Olympia oyster Trinity transcriptome aligned to genome with Bowtie2
Progress on generating bedgraphs from our Olympia oyster transcriptome continues.
Transcriptome Assembly - Olympia oyster RNAseq Data with Trinity
Used all of our current oly RNAseq data to assemble a transcriptome using Trinity.
DNA Methylation Analysis – Olympia Oyster Whole Genome BSseq Bismark Pipeline MethylKit Comparison
I previously ran two variations on the Bismark analysis for our Olympia oyster whole genome bisulfite sequencing data:
RNA Isolation - Lyophilized Tanner Crab Hemolymph in RNAlater
Due to difficulties getting RNA from hemolymph samples stored in RNAlater, Grace is testing out lyophilizing samples before extraction. Who knows what impact this will have on RNA, but it’s worth a shot!
DNA Methylation Analysis - Olympia Oyster Whole Genome BSseq Bismark Pipeline Comparison
Ran Bismark using our high performance computing (HPC) node, Mox, with two different bowtie2 settings:
DNA Quantification - Sea Lice DNA from 20180523
We previously received sea lice (Caligus tape) DNA from Cris Gallardo-Escarate at Universidad de Concepción.
DNA Methylation Analysis - Olympia oyster BSseq MethylKit Analysis
NOTE: IMPORTANT CAVEATS - READ POST BEFORE USING DATA.
Transcriptome Assembly - Geoduck RNAseq data
Used all of our current geoduck RNAseq data to assemble a transcriptome using Trinity.
FastQC/MultiQC/TrimGalore/MultiQC/FastQC/MultiQC - O.lurida WGBSseq for Methylation Analysis
I previously ran this data through the Bismark pipeline and followed up with MethylKit analysis. MethylKit analysis revealed an extremely low number of differentially methylated loci (DML), which seemed odd.
Transposable Element Mapping – Crassostrea virginica Genome, Cvirginica_v300, using RepeatMasker 4.07
Per this GitHub issue, I’m IDing transposable elements (TEs) in the Crassostrea virginica genome.
DNA Methylation Analysis - Bismark Pipeline on All Olympia oyster BSseq Datasets
Bismark analysis of all of our current Olympia oyster (Ostrea lurida) DNA methylation high-throughput sequencing data.
Data Received - Geoduck Metagenome HiSeqX Data
Received the data from the geoduck metagenome libraries that I prepared and were sequenced at the Northwest Genomics Center at UW on the HiSeqX (Illumina) - PE 151bp.
RNA Isolation & Quantificaiton - Tanner Crab Hemolymph
Isolated RNA from 40 Tanner crab hemolymph samples selected by Grace with the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s protocol, with the following modifications:
Bioanalyzer - Tanner Crab RNA Isolated with RNeasy Plus Mini Kit
Ran the four Tanner crab RNA samples that I isolated yesterday on the Seeb Lab Bioanalyzer 2100 (Agilent) using the RNA Pico 6000 Kit.
RNA Cleanup - Tanner Crab RNA
In a continued attempt to figure out what we can do about the tanner crab RNA, Steven tasked me with using an RNeasy Kit to cleanup some existing RNA.
RNA Isolation - Tanner Crab Hemolymph Using RNeasy Plus Mini Kit
Tanner crab RNA has proved a bit troublesome. As such, [Steven asked me to try isolating some RNA using the RNeasy Plus Mini Kit (Qiagen)(https://github.com/RobertsLab/resources/issues/327) to see how things would turn out.
Mox - Password-less SSH!
The high performance computing (HPC) cluster (called Mox) at Univ. of Washington (UW) frustratingly requires a password when SSH-ing, even when SSH keys are in use. I have a lengthy, unintelligible password that I use for my UW account, so having to type this in any time I want to initiate a new SSH session on Mox is a painful process.
Transposable Element Mapping – Olympia Oyster Genome Assembly, Olurida_v081, using RepeatMasker 4.07
I previously performed this analysis using a different version of our Ostrea lurida genome assembly. [Steven asked that I repeat the analysis with a modified version of the genome assembly (Olurida_v081)(https://github.com/RobertsLab/resources/issues/265#issuecomment-401055771) - only has contigs >1000bp in length.
Library Construction - Geoduck Water Filter Metagenome with Nextera DNA Flex Kit (Illumina)
Made Illumina libraries with goeduck metagenome water filter DNA I previously isolated on:
BS-seq Mapping – Olympia oyster bisulfite sequencing: Bismark Continued
Transposable Element Mapping – Crassostrea virginica NCBI Genome Assembly using RepeatMasker 4.07
Genome used: NCBI GCA_002022765.4_C_virginica-3.0
Read Mapping – Olympia oyster 2bRAD Data with Bowtie2 (on Mox)
Transposable Element Mapping - Olympia Oyster Genome Assembly using RepeatMasker 4.07
Software Installation – RepeatMasker v4.0.7 on Emu/Roadrunner Continued
After yesterday’s difficulties getting RMblast to compile, I deleted the folder and went through the build process again.
Software Installation - RepeatMasker v4.0.7 on Emu/Roadrunner
Steven asked that I re-run some Olympia oyster transposable elements analysis using RepeatMasker and a newer version of our Olympia oyster genome assembly.
TrimGalore/FastQC/MultiQC – TrimGalore! RRBS Geoduck BS-seq FASTQ data (directional)
Earlier this week, I ran TrimGalore!, but set the trimming, incorrectly - due to a copy/paste mistake, as --non-directional, so I re-ran with the correct settings.
FastQC - RRBS Geoduck BS-seq FASTQ data
Earlier today I finished trimming Hollie’s RRBS BS-seq FastQ data.
TrimGalore/FastQC/MultiQC – TrimGalore! RRBS Geoduck BS-seq FASTQ data
DNA Isolation & Quantification - Metagenomics Water Filters
After discussing the preliminary DNA isolation attemp with Steven & Emma, we decided to proceed with DNA isolations on the remaining 0.22μm filters.
Total Alkalinity Calculations - Yaamini’s Ocean Chemistry Samples
I ran a subset of Yaamini’s ocean chemistry samples on our T5 Excellence titrator (Mettler Toledo) at the beginning of April. The subset were samples taken from the beginning, middle, and end of the experiment. The rationale for this was to assess whether or not total alkalinity (TA) varied across the experiment. If there was little variation, then there’d likely be no need to run all of the samples. However, should there be temporal differences, then all samples should be processed.
TrimGalore/FastQC/MultiQC - Trim 10bp 5’/3’ ends C.virginica MBD BS-seq FASTQ data
Steven found out that the Bismarck documentation (Bismarck is the bisulfite aligner we use in our BS-seq pipeline) suggests trimming 10bp from both the 5’ and 3’ ends. Since this is the next step in our pipeline, we figured we should probably just follow their recommendations!
DNA Isolation & Quantification - Metagenomics Water Filters
Isolated DNA from the following two filters:
TrimGalore/FastQC/MultiQC - 2bp 3’ end Read 1s Trim C.virginica MBD BS-seq FASTQ data
Earlier today, I ran TrimGalore/FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch and hard trimmed the first 14bp from each read. Things looked better at the 5’ end, but the 3’ end of each of the READ1 seqs showed a wonky 2bp blip, so decided to trim that off.
TrimGalore/FastQC/MultiQC - 14bp Trim C.virginica MBD BS-seq FASTQ data
Yesterday, I ran TrimGalore/FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch with the default settings (i.e. “auto-trim”). There was still some variability in the first ~15bp of the reads and Steven wanted to see how a hard trim would change things.
TrimGalore/FastQC/MultiQC - Auto-trim C.virginica MBD BS-seq FASTQ data
Yesterday, I ran FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch. Steven wanted to trim it and see how things turned out.
FastQC/MultiQC - C. virginica MBD BS-seq Data
Per Steven’s GitHub Issues request, I ran FastQC on the Eastern oyster MBD bisulfite sequencing data we recently got back from ZymoResearch.
DNA Isolation & Quantification – Geoduck larvae metagenome filter rinses
This is another attempt to isolate DNA from two more of the geoduck hatchery metagenome samples Emma delivered on 20180313.
Titrations - Yaamini’s Seawater Samples
All data is deposited in the following GitHub repo:
Titrations - Yaamini’s Seawater Samples
All data is deposited in the following GitHub repo:
Data Received - Crassostrea virginica MBD BS-seq from ZymoResearch
Received the sequencing data from ZymoResearch for the Crassostrea virginica gonad MBD DNA that was sent to them on 20180207 for bisulfite conversion, library construction, and sequencing.
Titrations - Hollie’s Seawater Samples
All data is deposited in the following GitHub repo:
Titrations - Hollie’s Seawater Samples
All data is deposited in the following GitHub repo:
DNA Isolation & Quantification - Geoduck larvae metagenome filter rinses
Isolated DNA from two of the geoduck hatchery metagenome samples Emma delivered on 20180313 to get an idea of what type of yields we might get from these.
Titrations - Hollie’s Seawater Samples
Performed total alkalinity (TA) titrations on Hollie’s samples using our T5 Excellence titrator (Mettler Toledo) and Rondolino sample changer.
Titrations - Hollie’s Seawater Samples
Performed total alkalinity (TA) titrations on Hollie’s samples using our T5 Excellence titrator (Mettler Toledo) and Rondolino sample changer.
Progress Report - Titrator
I’ll begin this entry with a TL;DR (becuase it’s definitely a very long read):
Titrator Setup - Functional Methods & Data Exports
I’ve been working on getting our T5 Excellence titrator (Mettler Toledo) with Rondolino sample changer (Mettler Toledo) set up and operational.
Software Install - 10x Genomics Supernova on Mox (Hyak)
DNA Quantification - MspI-digested Crassostrea virginica gDNA
Quantified the two MspI-digested DNA samples for the Qiagen project from earlier today with the Qubit 3.0 (ThermoFisher).
Phenol:Chloroform Extractions and EtOH Precipitations - MspI Digestions of C.virginica DNA from Earlier Today
The two MspI restriction digestions from earlier today for our project with Qiagen were subjected to phenol:chloroform cleanup and subsequent ethanol precipitations.
Restriction Digestion - MspI on Crassotrea virginica gDNA
Digested two 1.5μg aliquots of Crassostrea virginica isolated 20171211, as part of the project we’re doing with Qiagen.
DNA Quantification - C.virginica MBD-enriched DNA
Quantified Crassostrea virginica MBD-enriched DNA from earlier today for Qiagen project.
MBD Enrichment – Crassostrea virginica Sheared DNA Day 3
Continued MBD enrichment of C.virginica DNA from yesterday for Qiagen project.
MBD Enrichment – Crassostrea virginica Sheared DNA Day 2
Continued MBD enrichment for C.virginica and Qiagen project from yesterday.
MBD Enrichment - Crassostrea virginica Sheared DNA Day 1
As part of a project with Qiagen to have them try out some of our DNA with their newest DNA bisulfite conversion kit, I previously [isolated DNA from Crassotrea virginica (Eastern oyster)(2017/12/11/dna-isolation-quantification-crassotrea-virginica-mantle-gdna.html) and sheared to ~420bp.
Tissue Sampling - Crassostrea virginica Tissues for Archiving
I figured it’d be prudent to collect some Eastern oyster (Crassotrea virginica) to have around the lab.
Software Install - MSMTP For Email Notices of Bash Job Completion on Emu (Ubuntu)
After I finally resolved the installation of PB Jelly on Emu (running Ubuntu 16.04), I’ve had a PB Jelly assembly running for the past two weeks! I’ve gotten tired of checking on its status (i.e. is it still running?) every day, so I dove in and figured out how to set up Emu to email me when the job is complete!
DNA Sonication & Bioanalzyer - C. virginica gDNA for MeDIP
I transferred 8ug (136uL) of Crassotrea virginica gDNA (isolated earlier today) to two separate 1.7mL snap cap tubes for sonication/shearing.
DNA Isolation & Quantification - Crassostrea virginica Mantle gDNA
DNA was isolated from a single adult Eastern oyster (Crassostrea virginica) for a pilot project with Qiagen to test their new DNA bisulfite conversion kit. The oyster was obtained yesterday afternoon (20171210) from the Taylo rShellfish Pioneer Square location. The oyster was stored @ 4C O/N.
Troubleshooting – PB Jelly Install on Emu Continued
The last “fix” didn’t fix everything.
Troubleshooting - PB Jelly Install on Emu
I previously installed and ran PB Jelly. Despite no error messages being output, I noticed something odd during my quick post-assembly stats check: The PB Jelly numbers were identical to the input reference file. This seemed very strange and made me decide to look a bit deeper in the PB Jelly output files.
DNA Isolation & Quantification - C. virginica Gonad gDNA
I isolated DNA from the Crassotrea virginica gonad samples sent by Katie Lotterhos using the E.Z.N.A. Mollusc Kit with the following modifications:
Software Installation - ALPACA on Roadrunner
List of software that needed installing to run ALPACA:
Software Crash - Olympia oyster genome assembly with Masurca on Mox
Ah, the joys of bioinformatics. I just received an email from Mox indicating that [the Masurca assembly I started 11 DAYS AGO (!!)(2017/10/19/genome-assembly-olympia-oyster-illumina-pacbio-reads-using-masurca.html) crashed.
Software Installation - PB Jelly Suite and Blasr on Emu
I followed along with what Sean previously did when installing on Emu, but it appears he didn’t install it in the shared location to make it accessible to all users. So, I’m installing it in the /home/shared/ directory.
Assembly Comparison - Oly PacBio Canu: Sam vs. Sean with Quast
I recently finished an assembly of our Olympia oyster PacBio data using Canu and thought it would be interesting to compare to Sean’s Canu assembly.
FAIL - Missing Data on Owl!
Uh oh. There appears to be some data that’s been removed from Owl. I noticed this earlier when trying to look at some of Sean’s data. His data should be in a folder with his name in Owl/scaphapoda
Software Installation - MaSuRCA v3.2.3 Assembler on Mox (Hyak)
Saw this tweet this morning and thought this would be good to try out for our Olympia oyster genome assemblies, as it will handle “hybrid” assemblies (i.e. short-reads and long reads):
Fail - Directory Contents Deleted!
Uh, not sure what happened here:
Genome Assembly - Olympia oyster PacBio minimap/miniasm/racon
In this GitHub Issue, Steven had suggested I try out the minimap/miniasm/racon pipeline for assembling our Olympia oyster PacBio data.
RNA Isolation - Olympia oyster gonad tissue in paraffin histology blocks
My previous go at this was a little premature - I didn’t wait for Laura to fully annotate her slides/blocks. Little did I know, the tissue was mostly visceral mass and, as such, I didn’t hit much in the way of actual gonad tissue. So, I’m redoing this, now that Grace has gone through and annotated the blocks to point out gonad tissue. SN-10-16 was sent to Katherine Silliman on 20170720.
RNA Isolation - Olympia oyster gonad tissue in paraffin histology blocks
UPDATE 20170712: The RNA I isolated below is from incorrect regions of tissue. I misunderstood exactly what this tissue was, and admittedly, jumped the gun. The tissue is actually collected from the visceral mass - which contains gonad (a small amount) and digestive gland (a large amount). The RNA isolated below will be stored in one of the Shellfish RNA boxes and I will isolate RNA from the correct regions indicated by Grace
Sample Annotation - Olympia oyster histology blocks (from Laura Spencer)
I’ve been asked to isolate RNA from some paraffin-embedded Olympia oyster gonad tissue.
Manuscript Re-submission - Oly Stress Response to PeerJ for Review
GitHub Curation
Updated a couple of GitHub Wikis:
DNA Methylation Quantification - Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)
Used the MethylFlash Methylated DNA Quantification Kit (Colorimetric) from Epigentek to quantify methylation in these coral DNA samples.
DNA Quantification - Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)
I quantified the three samples (listed below) that I SpeedVac’d yesterday using the the Roberts Lab Qubit 3.0.
DNA Concentration - Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)
Three samples (of the 62 total) that were quantified earlier today, had concentrations too low for use in the methylation assay:
DNA Quantification - Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)
DNA samples received yesterday were quantified using the Roberts Lab Qubit 3.0 to improve quantification accuracy (samples provided by Javier were quantified via NanoDrop, which generally overestimates DNA concentration) prior to performing methylation assessment.
Manuscript Writing - More “Nuances” Using Authorea
I previously highlighted some of the issues I was having using Authorea.com as an writing platform.
Data Received - Jay’s Coral RADseq and Hollie’s Geoduck Epi-RADseq
Jay received notice from UC Berkeley that the sequencing data from his coral RADseq was ready. In addition, the sequencing contains some epiRADseq data from samples provided by Hollie Putnam. See his notebook for multiple links that describe library preparation (indexing and barcodes), sample pooling, and species breakdown.
Manuscript Writing - The “Nuances” of Using Authorea
I’m currently trying to write a manuscript covering our genotype-by-sequencing data for the Olympia oyster using the Authorea.com platform and am encountering some issues that are a bit frustrating. Here’s what’s happening (and the ways I’ve managed to get around the problems).
Data Management - Geoduck RRBS Data Integrity Verification
Yesterday, I downloaded the Illumina FASTQ files provided by Genewiz for Hollie Putnam’s reduced representation bisulfite geoduck libraries. However, Genewiz had not provided a checksum file at the time.
Data Received - Geoduck RRBS Sequencing Data
Hollie Putnam prepared some reduced representation bisulfite Illumina libraries and had them sequenced by Genewiz.
Data Managment - Trim Output Cells from Jupyter Notebook
Last week I downloaded the final BGI data files and assemblies for Olympia oyster and geoduck genome sequencing projects. However, the output from the download command made the Jupyter Notebook files too large to view and/or upload to GitHub. So, I had to trim the output cells from that notebook in order to render it usable/viewable.
Computing - An Excercise in Futility
Trying to continue my Oly GBS analsyis from the other day and follow along with Katherine Silliman’s notebook
Data Received – Jay’s Coral epiRADseq - Not Demultiplexed
Previously downloaded Jay’s epiRADseq data that was provided by the Genomic Sequencing Laboratory at UC-Berkeley. It was provided already demultiplexed (which is very nice of them!). To be completionists on our end, we requested the non-demultiplexed data set.
Oyster Sampling - Olympia Oyster OA Populations at Manchester
I helped Katherine Silliman with her oyster sampling today from her ocean acidification experiment with Olympia oysters (Ostrea lurida) at the Kenneth K. Chew Center for Shellfish Research & Restoration, which is housed at the NOAA Northwest Fisheries Science Center at Manchester in a partnership with the [Puget Sound Restoration Fund (PSRF)(http://www.restorationfund.org/). We sampled the following tissues and stored in 1mL RNAlater:
Data Received - Jay’s Coral epiRADseq
We received notice that Jay’s coral (Porites spp) epiRADseq data was available from the Genomic Sequencing Laboratory at UC-Berkeley.
Manuscript Submission - Oly Stress Response to PeerJ for Review
Submitted the following manuscript to PeerJ for peer review:
Computing - Amazon EC2 Cost “Analysis”
I recently moved some computing jobs over to Amazon’s Elastic Cloud Computing (EC2) in attempt to avoid some odd computing issues/errors I kept encountering on our lab computers (Apple Xserve 3,1).
Computing - Not Enough Power!
Well, I tackled the storage space issue by expanding the EC2 Instance to have a 1000GB of storage space. Now that that’s no longer a concern, it turns out I’m running up against processing/memory limits!
Computing - A Very Quick “Guide” to Amazon EC2 Continued
Yesterday’s post ended with me trying to mount a S3 bucket to my EC2 instance using s3fs-fuse.
Dissection - Frozen Geoduck & Pacific Oyster
We’re working on a project with Washington Department of Natural Resources’ (DNR) Micah Horwith to identify potential proteomic biomarkers in geoduck (Panopea generosa) and Pacific oyster (Crassostrea gigas). One aspect of the project is how to best conduct sampling of juvenile geoduck (Panopea generosa) and Pacific oyster (Crassostrea gigas) to minimize changes in the proteome of ctenidia tissue during sampling. Generally, live animals are shucked, tissue dissected, and then the tissue is “snap” frozen. However, Micah’s crew will be collecting animals from wild sites around Puget Sound and, because of the remote locations and the means of collection, will have limited tools and time to perform this type of sampling. Time is a significant component that will have great impact on proteomic status in each individual.
Computing - The Very Quick “Guide” to Amazon Web Services Cloud Computing Instances (EC2)
This all takes a surprisingly long time to set up.
DNA Methylation Quantification - Coral DNA from Jose M. Eirin-Lopez (Florida International University)
Ran the coral DNA I quantified on 20160630 through the MethylFlash Methylated DNA Quantification Kit [Colorimetric] (Epigentek) kit to quantify global methylation.
DNA Quantification - Coral DNA from Jose M. Eirin-Lopez (Florida International University)
Quantified the DNA we received from Jose on 20160615 using the Qubit 3.0 Flouorometer (ThermoFisher) with the dsDNA Broad Range (BR) Kit according to the manufacturer’s protocol. Used 1μL of each sample.
Docker - One liner to create Docker container
One liner to create Docker container for Jupyter notebook usage and data analysis on roadrunner (Xserve):
GBS Frustrations
This isn’t really a notebook entry - it’s more of a traditional blog post.
Computing - Speed Benchmark Comparisons Between Local, External, & Server Files
I decided to run a quick test to see what difference in speed (i.e. time) we might see between handling files that are stored locally, on an external hard drive (HDD), or on our server (Owl).
Data Management - SRA Submission Overview
We have an enormous backlog of high-throughput sequencing files (641 FASTQ files, to be exact) that we need/want to get added to the NCBI Sequence Read Archive (SRA).
Data Management - High-throughput Sequencing Data
We’ve had a recent influx of sequencing data, which is great, but it created a bit of a backlog documenting what we’ve received.
PCR - Sea Pen luciferase
Ran a PCR to obtain luciferase DNA for sequencing.
Opticon2 Calibration
Jake and Steven recently noticed localized “hot spots” on most of Jake’s recent qPCRs, where higher levels of fluorescence were consistently showing up in interior portions of the plates than the outer portion of the plates.
Sample Submission – Olympia oyster PCRs Sanger Sequencing
Submitted a plate of purified PCR products (PCR products prepared by Jake on 20150623) that Jake set up yesterday, to the UW High-Throughput Genomics Center for Sanger sequencing.
Reverse Transcription - O.lurida DNased RNA Controls and 1hr Heat Shock
Performed reverse transcription on the Olympia oyster DNased RNA from the control samples and the 1hr heat shock samples of Jake’s project. To accommodate the large numbers of anticipated genes to be targeted in subsequent qPCRs, I prepared 100μL reactions (normally, 25μL reactions are prepared) using 250ng of each DNased RNA. A 1:10 dilution of the oligo dT primers (Promega) was prepared to improve pipetting accuracy. All incubations were performed in a thermal cycler without using a heated lid.
Sample Submission – Olympia oyster & Sea Pen PCRs Sanger Sequencing
Prepared two DNA plates and corresponding primer plates for sequencing at the UW HTGC from the purified gel-purified PCRs from yesterday. Primer plates were prepared by adding 7μL of NanoPure H2O to each well and then adding 3μL of 10μM primer to the appropriate wells. For the DNA plates, added 10μL of DNA to the appropriate wells.
Gel Purification - Olympia Oyster and Sea Pen PCRs
Purified DNA from the remaining PCR bands excised by Jake on 20150609 and 20150610, as well as Jonathan’s sea pen PCRs from 20150604, using Ultrafree-DA spin columns (Millipore). Transferred gel pieces from storage tubes (1.5mL snap cap tubes) to spin columns. Spun 10,000g, 5mins @ RT. Transferred purified DNA back to original storage tubes. See the sequence_log (Google Sheet) for a full list of the samples and the sequencing plates layouts. Purified DNA was stored @ 4C O/N. Will prepare and submit plates for Sanger sequencing tomorrow.
Sample Submission - Olympia oyster PCRs Sanger Sequencing
Submitted a plate of purified PCR products (PCR products prepared by Jake on 20150609 and 20150610) that Jake set up yesterday, to the UW High-Throughput Genomics Center for Sanger sequencing.
Reverse Transcription - Subset of Jake’s O.lurida DNased RNA
Currently don’t have sufficient reagents to perform reverse transcription on the entire set of DNased RNA (control and 1hr.heat-shocked O.lurida ctenidia samples). To enable Jake to start testing out some of his primers while we wait for reagents to come in, Steven suggested I generate some cDNA for him to use.
qPCR - Re-run Jake’s O.lurida DNased RNA Samples NC1, SC1, SC2, SC4 from 20150514
The following DNased RNA samples showed inconsistencies between qPCR reps (one rep showed amplification, the other rep did not) on 20150514:
qPCR – Jake’s O.lurida ctenidia DNased RNA (1hr Heat Shock Samples)
Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after the DNase treatment with Oly_Actin_F/R primers (SR IDs: 1505, 1504).
qPCR – Jake’s O.lurida ctenidia DNased RNA (Control Samples)
Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after the DNase treatment with Oly_Actin_F/R primers (SR IDs: 1505, 1504).
DNase Treatment - Jake’s O.lurida Ctenidia RNA (1hr Heat Shock) from 20150506
Since the O.lurida RNA I isolated on 20150506 showed residual gDNA via qPCR, I treated 1.5μg of RNA from each sample using the Turbo DNA-free Kit (Ambion/Life Technologies), following the “rigorous” protocol.
DNase Treatment - Jake’s O.lurida Ctenidia RNA (Controls) from 20150507
Since the O.lurida RNA I isolated on 20150507 showed residual gDNA via qPCR, I treated 5μg of RNA from each sample using the Turbo DNA-free Kit (Ambion/Life Technologies), following the “rigorous” protocol.
qPCR - Jake O.lurida ctenidia RNA (Heat Shock Samples) from 20150506
Ran qPCRs on the O.lurida total RNA I isolated on 20150506 to assess presence of gDNA carryover with Oly Actin primers (SR IDs: 1505, 1504).
qPCR - Jake O.lurida ctenidia RNA (Control Samples) From 20150507
Ran qPCRs on the O.lurida total RNA I isolated on 20150507 to assess presence of gDNA carryover with Oly Actin primers (SR IDs: 1505, 1504).
RNA Isolation – Jake’s O. lurida Ctenidia Control from 20150422
Isolated RNA from Jake’s Olympia oyster ctenidia, controls, collected on 20150422. Samples had been homogenized and stored @ -80C.
RNA Isolation - Jake’s O. lurida Ctenidia 1hr Heat Stress from 20150422
Isolated RNA from Jake’s Olympia oyster ctenidia, 1hr heat shock, collected on 20150422. Samples had been homogenized and stored @ -80C.
DNA Isolation - C.gigas Larvae from Katie Latterhos
Since the previous isolation attempt was unsuccessful (see 20140922), we’re trying a slightly different approach than yesterday.
DNA Isolation - C.gigas Larvae from Katie Latterhos and Emma
Isolated gDNA from two C.gigas larvae samples (stored in RNA Later) from Katie Latterhos:
PCR - Mac’s Bisulfite-Treated DNA
Realized that the PCR performed on 20140828 used the incorrect forward primer! As such, am repeating as before, but with the correct forward primer:
PCR - Mac’s Bisulfite-Treated DNA
Per Mac’s request, ran a PCR on a set of bisulfite-treated DNA (in her gDNA 2014 box in small -20C):
RNA Isolation - Jessica’s Geoduck Larval Stages
Isolated RNA from the following samples provided by Jessica Blanchette (stored in RNA later):
DNA Isolation - Mackenzie’s C.gigas EE2 Gonad Samples
Isolated DNA from the following samples, provided by Mackenzie:
DNA Isolation - Test Sample
Due to the recent poor quality gDNA that has been isolated from C.gigas gonad, I decided to do a quick test using TE for DNA pellet resuspension in hopes that old Buffer EB (Qiagen) or old nuclease-free H2O (Promega) are to blame for the apparent, rapid degradation that I’ve experienced.
DNA Isolation - Mackenzie’s C.gigas Gonad Sample
Mac’s been having some difficulty getting good quality gDNA from some of her gonad samples, so she asked me to give it a shot.
DNA Quality Check - Yanouk’s Oyster gDNA
We’ve had some Illumina sequencing issues with Yanouk’s samples, so I ran the samples out on a 0.8% agarose gel to evaluate the levels of degradation. Loaded 2uL of each sample. Did not load equal quantities of gDNA, due to the lack of available gDNA in the samples we submitted for Illumina sequencing. Added 2uL of H2O to samples 37 & 38 in hopes of having sufficient DNA for visualization on the gel.
DNA Isolation - Geoduck
Isolated additional geoduck gDNA from the two fresh (now frozen) geoduck’s that Brent provided me with on 20140212 so that we can potentially isolate RNA from the same geoducks to tie in with the DNA Illumina sequencing that Axa will be conducting. Isolated DNA using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol (incubated minced siphon tissue at 56C for 3hrs). Eluted with 75uL of ddH2O and spec’d on NanoDrop1000.
DNA Precipitation - Geoduck DNA from 20140213
After speaking with Axa regarding the DNA concentrations, he would like the DNA from the ethanol-fixed tissue to be more concentrated, and he wants them in ddH2O instead of Buffer AE (from the Qiagen DNeasy Kit). So, I preformed a standard ethanol precipitation. Added 0.1 volumes of 3M sodium acetate (pH = 5.2) [15uL], 2.5 volumes of 100% ethanol [412.5uL] and incubated @ -20C over the weekend.
DNA Isolation - Geoduck
Since yesterday’s DNA isolation failed to yield sufficient quantity of DNA from the ethanol-fixed samples, I isolated additional DNA from the same samples.
DNA Isolation - Geoduck
Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:
Gylcogen Assay - Emma’s C.gigas Whole Body Samples (continued from yesterday)
Samples from yesterday were centrifuged 30mins, 4000g, 4C (fixed angle rotor).
Gylcogen Assay - Emma’s C.gigas Whole Body Samples
Finally located the remaining half of Emma’s samples. These had already been freeze dried AND pulverized! So, I just had to weigh out ~half of each sample for the glycogen assay.
Gylcogen & Carboyhydrate Assays - Emma’s C.gigas Whole Body Samples (continued from yesterday)
Samples from yesterday were centrifuged 30mins, 4000g, 4C (fixed angle rotor).
Gylcogen and Carboyhydrate Assays - Emma’s C.gigas Whole Body Samples
Samples were previously freeze dried overnight and stored @ -20C. To maximize sample homogeneity and, thus, increase accuracy of both assays, all samples were mechanically pulverized in their existing tubes. Approximately half of each sample was weighed and used for the glycogen assay. The remainder of each sample was stored @ -20C.
PCR - Lake Trout C1q
PCR - Lake Trout C1q
DNA Quantification - Yanouk’s DNA Samples
PCR - Lake Trout C1q
Ran PCR on lake trout DNA and lake trout bisulfite-converted DNA. Used primers SRIDs: 1551 and 1552. DNA was isolated by Caroline Storer on 4/4/2011 and bisulfite converted on 4/7/2011. Master mix and cycling params are here:
PCR - Hexokinase Partial CDS
Performed PCR using the primers CG_HK_CDS_2132-2158 (SRID: 1521) and Cg_Hk_CDS_3’_no_stop (SRID: 1519) on pooled C.gigas cDNA (from DATE).
PCR - Hexokinase and Partial Exon #1
Performed PCR using newly designed primers to amplify the C. gigas hexokinase “promoter” (-2059bp from start) along with a portion of the first exon.
PCR - Hexokinase Promoter and CDS (repeat from 20130227)
Performed a repeat of the failed PCR from 20130227, but used a pool of cDNA (made from 20110311 C.gigas cDNA) instead of a single sample and changed the annealing temp to 50C.
PCR - Hexokinase Promoter and CDS
Performed PCR to amplify the C.gigas hexokinase (ACCESSION#) promoter region (-2059bp) and the CDS without the stop codon. Elimination of the stop codon allows for subsequent cloning into the pBAD-TOPO expression vector, which will incorporate the V5 epitope tag sequence. This tag will be used to distinguish between endogenous hexokinase expression and expression generated from our hexokinase construct.
Reverse Transcription - Herring RNA from 20091026
Performed an RT reaction on pooled herring gonad and liver mRNA from 20091026 for James Raymond at the UNLV. A single RT reaction was performed using 12.75uL (208ng) of the pooled gonad mRNA and 5uL (132.5ng) of the pooled liver RNA, according to our default MMLV (Promega) protocol. After reaction was completed, sample was stored @ -20C and then shipped to James Raymond on 20130214.
qPCR - Halley cDNA Check
Ran qPCR on Halley’s cDNA to see if I could get them to work. She has been getting high levels of fluorescence at the initiation of the qPCR cycling that shouldn’t be there. Master mix calcs and plate layout can be seen here. http://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20121128%20qPCR%20Layout.jpg
Reverse Transcription - FISH441 RNA
Reverse transcribed the class FISH441 RNA to cDNA. Followed protocol provided on the FISH441 Wiki page, NOT the usual Roberts Lab protocol, with some modifications.
qPCR - Manila Clam Larvae cDNA (from August 2012 - Dave’s Notebook)
Ran qPCR on manila clam larvae cDNA that Dave made on 8/7/2012, using the sample sets from 7/29/2011 and 8/5/2011 of the OA manila clam experiment he ran.
qPCR - Manila Clam Larvae cDNA (from August 2012 - Dave’s Notebook)
Ran qPCR on manila clam larvae cDNA that Dave made on 8/7/2012, using the sample sets from 7/29/2011 and 8/5/2011 of the OA manila clam experiment he ran.
qPCR - Manila Clam Larvae cDNA (from August 2012 - Dave’s Notebook)
Ran qPCR on manila clam larvae cDNA that Dave made on 8/7/2012, using the sample sets from 7/29/2011 and 8/5/2011 of the OA manila clam experiment he ran.
qPCR - DNased Manila Clam Larvae RNA (from August 2012 - Dave’s Notebook)
Performed qPCR on Dave’s manila clam larvae DNased RNA from August 2012 using EF1a primers (SR IDs: 1463, 1474).
Received oysters from Taylor Shellfish.
Photos of oysters are here:
qPCR - Opticon Test
Ran qPCR to test uniformity of Opticon 2, after it was serviced on 20120926.
Minipreps - Emma’s Illumina Library Cloning
Performed plasmid isolation on 17 bacterial cultures Emma inoculated yesterday using the QIAprep Spin Mini Kit (Qiagen) using ~1.4mL of culture according to the manufacturer’s protocol. Plasmid DNA was eluted with 50uL of Buffer EB. Tubes were stored @ 4C in the refrigerator in FTR 213.
Illumina RNAseq Library Construction - 32 C.gigas Individuals
Took heat-fragmented RNA provided by Emma (see Emma’s Notebook, 7/3/2011) and proceeded to make first strand cDNA, as described in the Eli Meyer protocol for Illumina HiSeq. Master mix calcs are here. Samples were stored @ -20C after the reverse transcription and library construction will be continued tomorrow.
Oligo Reconstitution - Illumina RNAseq Library Oligos and Barcodes
Reconstituted all of the oligos and barcodes for library construction in TE (pH = 8.0) to a final concentration of 100uM. Created 10uM working stocks of all oligos and barcodes. All samples (stocks and working stocks) are stored @ -80C in their own box (Illumina Library Oligos & Barcodes) due to the fact that one of the oligos is an RNA oligo and requires storage at -80C.
Chloroform Clean Up - Lexie’s QPX RNA from 20110504
After submission of QPX samples to HTGU for Illumina library prep yesterday, I was notified that there was insufficient RNA for the QPX RNA samples. I checked the source RNA on the Roberts Lab NanoDrop1000 and determined that they had high phenol contamination (large peak at 270nm), which results in a large exaggeration in the OD260 absorbance (NanoDrop1000 report[JPEG]; notice terrible OD260/280 ratios; did not save screen shot of absorbance peaks.). As such, the concentrations that Lexie had listed in her notebook for these samples are highly inaccurate and highly inflated. To remove the phenol, I brought all of her QPX RNA samples from 20110504 up to ~200uL with 0.1%DEPC-H2O, added 200uL of chloroform, vortexed for 30s, spun at 12,500g RT for 15mins, and transferred aqueous phase to new tube. Then performed an ethanol precipitation on the aqueous phase. Added 0.1 vols of 3.0M sodium acetate (pH = 5.2), 2.5 vols of 100% EtOH, mixed and incubated at -20C for 1hr. Pelleted RNA by spinning at 16,000g 4C for 15mins.
Sample Submission - QPX RNA and DNA for Illumina 36bp single-end RNA/DNAseq
Submitted samples to HTGU for Illumina sequencing.
QPX Sample Pooling for Illumina Sequencing
Pooling volumes/quantities for QPX RNA/DNA-seq: 20120705 Oly larvae and QPX HiSeq Calcs
qPCR - Detection of V.tubiashii Presence and Expression Using VtpA Primers in DNA/cDNA from yesterday
Ran qPCR with VtpA primers on cDNA and DNA (from yesterday) of C.gigas larvae to see levels of V.tubiashii compared to their water filter samples (see 20120326). Master mix calcs are here. Plate layout, cycling params, etc can be seen in the qPCR Report (see Results). Used 1uL of cDNA and 100ng (1uL) of DNA as template.
Reverse Transcription - DNased C.gigas Larval RNA from 20120427
Performed reverse transcription using random primers (Promega) diluted 1:100 (5ng/uL) with 175ng of DNased total RNA. Random primers were used because we will be targeting V.tubiashii RNA instead of eukaryotic RNA. Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s protocol. Calcs are here.
qPCR - Check DNased RNA from Earlier Today for Residual gDNA
Ran qPCR using V.tubiashii VtpA primers (from Elene; no SR ID). Used 0.5uL of each DNased RNA sample, which equals ~40ng of RNA, which would be the equivalent amount of RNA that would end up in a qPCR rxn after cDNA has been made (using 1uL of cDNA). Used the filter DNA extraction from samples #279 from DATE as a positive control. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).
DNase Treatment - C.gigas Larvae RNA from yesterday
Treated 5ug of total RNA (in a 50uL rxn) using Turbo DNA-free (Ambion) according to the “Standard” protocol. Samples were spec’d on the Roberts Lab NanoDrop 1000.
gDNA Isolation - C.gigas Larvae from Taylor Summer 2011
Samples that had been split from earlier today (see the RNA Isolation below) were resuspended in 1mL of DNAzol (MRC). 100ug of Proteinase K (Fermentas) was added to each sample. Samples were incubated at RT, O/N on a rotator. On 20120427 samples were pelleted 10mins, 10,000g, and supe transferred to fresh tube. DNA was precipitated with 0.5mL of 100% EtOH, mixed gently and pelleted 5mins, 5000g. Supe was discarded, pellets were washed with 1mL 75% EtOH, re-pelleted at same speed as previous step, supe discarded and pellets were resuspended in NanoPure H2O. Samples were spec’d on the Roberts Lab NanoDrop1000.
RNA Isolation - C.gigas Larvae from Taylor Summer 2011
Samples had been stored in RNA Later (Ambion). Samples were pelleted and the RNA Later supe removed. Samples were washed (2x) with 1mL TE (pH = 8.0) to remove excess salt resulting from the RNA Later. Samples were split, roughly equally, into two separate tubes. Samples were pelleted and the supe removed. One tube from each sample was set aside for gDNA isolation using DNAzol (MRC). The other tube was vortexed vigorously in TriReagent (MRC) and the then treated according to protocol. Samples were resuspended in 100uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab NanoDrop 1000.
qPCR - Taylor Water Filter DNA Extracts from 20120322
Ran qPCR on the Taylor water filter DNA extracts from 20120322 using V.tubiashii VtpA primers (provide by Elene; no SR ID?) instead of 16s primers, which failed to produce acceptable results in the melt curves (see 20120323). Additionally, Elene has a standard curve for V. tubiashii (from 1/12/2011) based off of CFUs/mL, which will allow us to quantify theoretical number of V.tubiashii CFUs present in each sample.
qPCR - Repeat of qPCR from Earlier Today
Repeated exactly what was done earlier today due to apparent contamination in negative controls.
qPCR - Taylor Water Filter DNA Extracts from Yesterday
Ran qPCR on the Taylor water filter DNA extracts from yesterday using V.tubiashii 16s primers (SR IDs: 455, 456). Used RE22 DNA as a positive control, provided by Elene. Master mix calcs are here. All samples were run in duplicate. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).
DNA Extraction - Taylor Water Filter Samples from 2011
Extracted DNA from the following water filter samples using the Qiagen DNeasy Blood & Tissue Kit:
Reverse Transcription - Dave’s Manila Clam (Venerupis philippinarum) DNased RNA from 20120307 and 20120302
Performed reverse transcription on 1.5ug of DNased RNA in a 75uL reaction, using oligo dT primers. All reagents were scaled appropriately (based on Promega’s M-MLV RT protocol). Samples were prepared in a plate and stored @ -20C. Plate layout and all reverse transcription calcs are here:
qPCR - Dave’s Manila Calm (Venerupis philippinarum) DNased RNA from yesterday and 20120302
Performed qPCR on all DNased RNA samples from this group (samples #1-48) using beta actin primers (SR IDs: 1379, 1380). 0.5uL of each DNased RNA was used, which was the equivalent of ~40ng, in order to simulate the amount of RNA present in the subsequent cDNA (1000ng of RNA in 25uL cDNA; use 1uL of cDNA in qPCR reaction). Master mix calcs are here. Plate layout, cycling params, etc., can be found in the qPCR Report (see Results). 0.5uL of total RNA from sample Vp gill 01 was used to serve as a positive control, since Dave has no existing V. phillippinarum cDNA.
RNA Isolation - Dave’s Manila Clam (Venerupis philippinarum) Gill Samples (#25-48)
Isolated RNA from Manila Clam gill samples provided by Dave, according to protocol. Samples were resuspended in 0.1%-DEPC H2O and spec’d on the Roberts Lab NanoDrop1000. Samples were stored @ -80C in Dave’s box that the tissue was initially stored in.
DNase - Dave’s Manila Clam (Venerupis philippinarum) Gill RNA from Yesterday
DNased RNA using Ambion’s Turbo DNA-free Kit following the “routine” protocol. 5ug of total RNA from each sample was treated in 50uL reactions. Samples will be spec’d on Monday with the Roberts Lab NanoDrop 1000.
RNA Isolation - Dave’s Manila Clam (Venerupis philippinarum) Gill Samples (#1-24)
Isolated RNA from Manila Clam gill samples provided by Dave according to protocol. Samples were resuspended in 0.1%-DEPC H2O and spec’d on the Roberts Lab NanoDrop1000. Samples were stored @ -80C in Dave’s box that the tissue was initially stored in.
qPCR - cDNA from 20120208
Performed qPCR on all 12 samples. Used the following primers, provided by Elene, to detect V.tubiashii expression:
qPCR - cDNA from earlier today
Performed qPCR on all 12 samples. Used Cg_EF1aF/R2 (SR IDs: 1410 & 1412) for one set of qPCRs and Vtub_16s_F/R (SR IDs: 455 & 456) for the other set of qPCRs. Used pooled C.gigas cDNA (from 20110311) and RE22 DNA (provided by Elene) as positive controls for C.gigas and V.tubiashii, respectively. C.gigas gDNA (7ng of BB16 from 20110201) was used as a negative control for EF1a. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). All samples were run in duplicate.
Reverse Transcription - C.gigas larvae DNased RNA (from 20120125)
Performed reverse transcription on Dnased RNA from 20120125 using 175ng RNA from each sample. Also used random primers (instead of the usual Oligo dT primers) since these samples will also be used to analyze gene expression in Vibrio tubiashii. cDNA calcs are here.
qPCR - DNased RNA from earlier today
Checked DNased RNA samples from earlier today for the presence of residual gDNA. Used C.gigas BB16 gDNA (from 20110201) diluted to ~7ng/uL as a positive control to match the dilution factor of the RNA that will be used in the reverse transcription reaction (175ng in 25uL = 7ng/uL). All samples were run in duplicate. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).
DNAse - C.gigas RNA from 20120124
5ug of each RNA was DNased using Ambion’s Turbo DNA-free Kit, according to the rigorous protocol and spec’d on Roberts Lab NanoDrop 1000. RNA volume calcs are here.
RNA Isolation - C.gigas Larvae from 20110412 & 20110705 (Continued from 20120112)
All of the RNA samples were re-combined with their respective counterparts and subject to a standard EtOH precipitation (0.1 volumes of 3M NaOAc, pH = 5.2, 2.5 volumes 100% EtOH; incubated -80C 1hr; pelleted; washed with 1mL 70% EtOH; pelleted). Pellets were washed two additional times (for a total of three washes) with 70% EtOH. RNA was resuspended in 50uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab NanoDrop 1000.
RNA Isolation - C.gigas Larvae from 20110412 & 20110705
RNA was isolated from C.gigas larvae collected from Taylor Shellfish hatchery on the dates noted above. Samples were in RNA Later. RNA Later was removed. Attempted homogenization with a pestle proved futile, as a significant quantity of larvae were sticking to the pestle and were nearly impossible to wash off using TriReagent as a rinsing agent. Due to this, all samples were vortexed for 1min in 1mL of TriReagent. It should be noted that the TriReagent took on a cloudy appearance and even showed some separation into two layers upon letting the samples sit. This was not normal and I was immediately concerned about the high salt content from residual RNA Later. Samples were treated normally with the following changes:
Sequencing - COX/PGS Clones from yesterday/today
Samples were submitted for sequencing to the University of Washington HTGU, two times from each direction using vector M13F/R primers. See Sequence Log for plate layout.
Mini-preps - COX/PGS Cloning Colonies from today
Selected 10 colonies (1-8, 18, 28) for mini-preps. Inoculated 5mL 1x LB + 50ug/mL of Kanamycin. Incubated O/N, 37C, 200RPM. 3mL of each culture were used for mini-preps. Used Qiagen kit. Samples were eluted w/30uL of EB.
PCR - COX/PGS Cloning Colony Screens from yesterday
Performed PCR on 40 colonies using both qPCR primer sets to see if I could differentiate between which colonies potentially contained each isoform to reduce the amount of clones needed for sequencing. Master mix and cycling params are here. Primers used were:
Cloning - Purified COX/PGS “qPCR Fragment” from 20111006
Cloned the purified “qPCR Fragment” from 20111006 using the TOPO TA Cloning Kit (Invitrogen). Performed a half reaction (total volume = 3uL), using 1uL of purified PCR product. Incubated at RT for 20mins and then placed reaction on ice. Transformed chemically competent TOP 10 cells (Invitrogen) and heat shocked at 42C for 30s. Added 250uL of RT S.O.C. medium and incubated at 37C, 200RPM. Plated cells on pre-warmed Kan50+X-Gal plates (plates from 20110726; X-Gal added ~30mins before plating cells). Incubated plates O/N, 37C.
PCR - Purified COX/PGS 1/2 DNA from earlier today
Ran PCR using primers Cg_COX1/2_qPCR_F, Cg_COX1_qPCR_R, Cg_COX2_454align1_R (SR IDs: 1192, 1191, 1190; respectively). Template was pooled cDNA from 20110311 of various C.gigas tissues. These reactions will verify (sort of) if we have both isoforms present in the PCR performed earlier today, prior to cloning. Master mix calcs and cycling params are here.
PCR - Region Outside of COX/PGS qPCR Primers
Ran PCR using primers Cg_COX_982_F and Cg_COX_2138_R (SR IDs: 1149 & 1151, respectively). Template was pooled cDNA from 20110311 of various C.gigas tissues. These primers anneal 5’ and 3’ of where the qPCR primers for both COX1/PGS1 and COX2/PGS2 anneal. Master mix calcs and cycling params are here. Ran multiples of the same reaction to ensure sufficient product for use in cloning/PCR.
Ethanol Precipitation - Full-length PGS1 cDNA (from 20110921)
Performed an EtOH on gel-purified PCR products from 20110921. Briefly, added 0.1 vols of 3M sodium acetate (pH=5.2; 43uL), mixed and then added 2.5 vols of 100% EtOH (1182.5uL). Mixed, split into two tubes (due to high volume not fitting in a single tube) and incubated @ -80C O/N. Pelleted DNA 16,000g, 20mins, 4C. Discarded supe. Washed pellet w/ 1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Resuspended both pellets in a TOTAL of 25uL Qiagen Buffer EB (10mM Tris-HCl) and spec’d.
PCR - Full-length PGS1 cDNA
Still have insufficient quantities of DNA for sequencing. Master mix calcs and cycling params are here. Additionally, used some of the purified PCR product as template in one of the reactions, just for comparison purposes. cDNA template was pooled cDNA from 20110311 from various C.gigas tissues. Also, increased the amount of template 4-fold in an attempt to obtain higher yields of PCR products for sequencing.
Ethanol Precipitation - Purified PGS1 PCR from yesterday
Added 0.1 vols of 3M sodium acetate (pH = 5.2; 38uL) and 2 vols of 100% EtOH (836uL). Incubated 30mins @ -20C. Pelleted DNA 16,000g, 15mins, 4C. Removed supe. Washed pellet w/1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Removed supe. Air-dried pellet. Resuspended in 50uL of Qiagen EB Buffer and spec’d.
PCR - Full-length PGS1 cDNA
Need more PCR product for sequencing. Repeated reaction from 20110825.
PCR - Full-length PGS2 cDNA
Repeated PCR from 20110825 to attempt to amplify the full-length cDNA for PGS2 (COX2), however this time using a more robust polymerase (Amplitaq Gold) in hopes of getting results. Additionally, tried 3 different Mg2+ concentrations (1.5mM, 2.0mM, and 3.0mM). Master mix calcs and cycling params are here. cDNA was pooled cDNA made 20110311 from various tissues. PGS2 primers = 1376, 1375.
PCR - Full-length PGS1 & PGS2 cDNAs
Ran PCR to amplify full-length cDNAs of PGS1 & PGS2 (COX1 & COX2) using primers designed to anneal in the 5’/3’UTRs of each isoform. PGS1 primers = SRIDs: 1377, 1378. PGS2 primers = 1376, 1375. Master mix calcs and cycling params are here. cDNA was pooled cDNA made 20110311 from various tissues.
qPCR - C.gigas V.vulnificus Exposure cDNA (from 20110311)
Ran a qPCR using 3hr Vibrio vulnificus exposure cDNA from 20110311. Original experiment conducted on 20110111 with defensin primers (SR IDs: 1109 & 1070) and GAPDH (SR IDs: 1172 & 1173). Master mix calcs are here. Cycling params, plate layout, etc can be seen in the qPCR Report (see Results). This was performed to help Herschel.
Sequencing - C.gigas COX2/PGS2 Clone #4 from 20110728
Used new primers for sequencing (SR IDs: 1351 & 1352) clone #4. Sequenced clone two times in each direction. DNA and primers were sent for sequencing at ASU. Requested “High GC” treatment to help overcome the issue seen on 20110728.
Plasmid Isolation & Sequencing - C.gigas COX2/PGS2 Clones (from yesterday)
Isolated plasmid DNA from 3mL of liquid cultures that were inoculated yesterday using Qiagen’s miniprep kit. DNA was eluted with 50uL of EB. DNA was prepped and sent for sequencing to ASU sequencing facility. Each clone was sequenced two times in each direction. Samples are as follows:
Bacterial Cultures - C.gigas COX2/PGS2 Clones (from yesterday)
Inoculated 4 x 5mL 1xLB + Kan50 with a colony from each set of clones, incubated 37C, 200RPM, O/N. Will mini prep and send for sequencing tomorrow.
Colony PCRs - C.gigas COX2/PGS2 Clones (from yesterday)
Performed colony PCRs on the 4 sets of cloning reactions that were performed yesterday using the M13F/R vector primers. Colonies were picked, restreaked on a fresh LB Kan50 plates (made 20110726 by SJW) and PCR’d. Master mix calcs are here. Selected 8 white colonies from each cloning reaction for PCR. Restreaked plate was incubated @ 37C O/N.
Cloning - C.gigas COX2/PGS2 5’/3’ RACE Products (from earlier today)
The bands that were excised and purified earlier today were cloned in to pCR2.1 using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer’s protocol with the following changes: used 4uL of all PCR products, incubated ligation reactions for 15mins @ RT, incubated competent cells with ligation reactions for 15mins on ice.
5’/3’ RACE - C.gigas COX2/PGS2 Nested RACE PCR
Performed nested RACE PCR on the RACE PCR products generated on 20110722 using the following nested primers: PGS2_ngspRACE_5’ (SR ID: 1350) and PGS2_ngspRACE_3’ (SR ID: 1349). Removed 2uL from each of the primary PCR reactions and brought up to 100uL in tricine EDTA (supplied in the Clontech SMARTer RACE cDNA Amplification Kit). Performed the nested RACE PCR according to the Clontech manual. Briefly, this is the same as the primary RACE PCR reaction, but using 5uL of the diluted primary PCR product and 1uL of the Nested Universal Primer (instead of 5uL of the 10X Universal Primer Mix). Master mix calcs and set up are here. Cycling params followed “Program 2” of the Clontech protocol, modified for nested primers, and are as follows:
5’/3’ RACE - C.gigas COX2/PGS2 RACE PCR
Additional RACE using gene specific primers (SR IDs: 1347 & 1348) for C.gigas COX2/PGS2 according to Clontech’s SMARTer cDNA RACE Kit protocol. 3’/5’ RACE cDNA libraries are from 20080619. Master mix calcs and set up is here. Cycling params followed “Program 2” of the Clontech protocol and are as follows:
Sequencing - PGS Hi 4 (PGS2/COX2)
Sent plasmid prep to ASU (5uL of plasmid + 1uL of 10uM M13F/R). SJW01 = M13F, SJW02 = M13R.
Plasmid Isolation - Miniprep on PGS Hi 4 Colony from yesterday
Plasmid was isolated from 3mL of liquid culture started yesterday using the Qiagen MiniPrep Kit, according to the manufacturer’s protocol. Will send off for sequencing.
PCR - Colony PCR on Restreaked PGS2 Clones from 20110707
Ran a colony PCR at the same time that I inoculated liquid cultures, using M13 primers.
Bacterial Cultures - Liquid Cultures of PGS2/COX2 Colonies from 20110707
Clone Restreaking - PGS2 Hi/Lo Clones (from 20110421)
Sequencing of the PGS2/COX2 clone failed (was empty vector). Restreaked bacterial clones on to a Kan50 plate (made 20110413 by SJW) from a plate that Caroline Storer had created from cloning colony selection on 20110421. Samples were labeled as PGS Lo 1 & 2 and PGS Hi 3 & 4. Additionally, there were red numbers on the plate associated with these four samples. They were 42 - 45, respectively. PGS Hi 4 (#45) was previously grown up and sequenced. This sample is what produced vector only sequence. Incubated O/N @ 37C. Hopefully we’ll bacteria is still viable and will have samples to grow up for miniprep, plamsid iso and sequencing.
qPCR - C.gigas GAPDH second rep on V.vulnificus exposure cDNA (from 20110311) and standard curves for COX1, COX2, GAPDH
Ran a qPCR on all cDNA samples. Created a standard curve to possibly allow for use of the BioRad software for gene expression analysis. Standard curve was created from pooled cDNA (1uL from each individual sample). Master mix calcs are here.
qPCR - C.gigas actin and GAPDH on V.vulnificus exposure cDNA (from 20110311)
Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. This qPCR was to test 2 of 4 potential normalizing genes to evaluate which genes show the least amount of effect from the treatments in this experiment. Primers for actin used were Cg_Actin_306_F (SR ID: 1170), Cg_Actin_408_R (SR ID: 1171). Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).
qPCR - C.gigas 18s and EF1a on V.vulnificus exposure cDNA (from 20110311)
Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. This qPCR was to test 2 of 4 potential normalizing genes to evaluate which genes show the least amount of effect from the treatments in this experiment. Primers for 18s used were Cg_18s_1644_F (SR ID: 1168), Cg_18s_1750_R (SR ID: 1169). Primers for EF1a used were EF1_qPCR_5’ (SR ID: 309), EF1_qPCR_3’ (SR ID: 310)Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).
qPCR - C.gigas COX2 on V.vulnificus exposure cDNA (from 20110311)
Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. Primers used were Cg_COX1/2_qPCR_F (SR ID: 1192) & Cg_COX2_454align1_R (SR ID: 1190). Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).
qPCR - C.gigas COX1 on V.vulnificus exposure cDNA (from 20110311)
Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. Primers used were Cg_COX1/2_qPCR_F (SR ID: 1192) & Cg_COX1_qPCR_R (SR ID: 1191). Samples were run in duplicate. Master mix calcs are here. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).
qPCR - Hard Clam NGS Primer Checks
Ran a qPCR to evaluate a large batch of primers (40 sets) that were ordered per Steven, based off of the most recent SOLiD run (samples submitted 3/10/2011; see Dave’s notebook for more info). Pooled cDNA (2uL from each individual; from 20110511) was used. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). The list of primers tested is available in the Primer Database and consist of SR IDs 1233 - 1312. For brevity, samples were only labelled with the corresponding contig number.
qPCR - Lexie’s QPX Temp & Tissue Experiment (see Lexies Notebook 4/26/2011)
Ran qPCR with Lexie’s cDNA samples from this experiment with the following primer sets in order to better evaluate her biological reps:
qPCR - Emma’s New 3KDSqPCR Primers
Due to previous contamination issues with Emma’s primers, Emma asked me to order new primers, reconstitute them and run a qPCR for her to see if we could eliminate her contamination issues with this primer set. cDNA template was supplied by Emma (from 2/2/11) and was from a C.gigas 3hr Vibrio vulnificus challenge. Samples were run in duplicate, as requested. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). Primer set used was:
Reverse Transcription - Hard Clam Gill DNased RNA (from 20110509)
Performed reverse transcription on DNased RNA from the hard clam vibrio tubiashii challenge experiment (see Dave’s Notebook 5/2/2011), following the Promega M-MLV RT protocol with ~1ug of DNAsed RNA. Master mix calcs are here. Reactions were done in a plate. cDNA was diluted 1:4 with H2O.
DNase - Hard Clam Gill RNA (from earlier today)
DNased 5ug of RNA from each sample with Ambion’s Turbo DNA-free Kit, according to the manufacturer’s rigorous protocol. Samples were spec’d and stored @ -80C in the “Hard Clam V.t. Experiment RNA” box.
RNA Isolation - Hard Clam Gill Tissue from Vibrio Experiment (see Dave’s Notebook 5/2/2011)
Isolated RNA in 1mL of Tri-Reagent according to the manufacturer’s protocol. Also, finished RNA isolation of samples that were started 20110506. Samples were resuspended in 50uL 0.1%DEPC-H2O and spec’d.
Primer Design - Hard Clam NGS Primers
Designed primers for 40 PCR targets derived from the most recent SOLiD data by Steven(Evernote link) using BatchPrimer3. BatchPrimer3 results are here.
RNA Isolation - Hard Clam Gill Tissue from Vibrio Experiment (see Dave’s Notebook 5/2/2011)
Isolated RNA in 1mL of Tri-Reagent according to manufacturer’s protocol. Samples were precipitated with isopropanol and stored over the weekend @ -20C. Will conclude isolation on Monday. The samples isolated were:
Received - Live Hard Clams From Scott Lindell
Received two bags containing ~24 live clams (didn’t count) in each bag. One bag labeled as “Mashpee Control” and the other “BX Selected.” Clams were stored @ 4C by Steven.
Bacterial Cultures - Colonies Selected from Yesterday’s Colony PCRs
Inoculated 5mL of 1x LB + Kan50 (made by Steven on 3/23/11). Incubated O/N at 37C, 250RPM. Will perform mini preps tomorrow. The following samples were selected:
Mini Preps - Liquid Cultures from yesterday
Mini prepped 3mLs of each culture, according to Qiagen protocol. Samples were eluted with 50uL of Buffer EB.
Bacterial Cultures - Colonies Selected by Steven from Steven’s Re-Streaked Plate
Inoculated 5mL of 1x LB + Kan50 (made by Steven on 3/23/11). Incubated O/N @ 37C, 250RPM. Will perform mini preps tomorrow. The following samples were selected (red text on the plate):
Colony PCR - Colonies from COX1 Genomic Cloning (from 20110411)
Ran colony PCR on various colonies produced from cloning on 20110411. All colonies were picked, re-streaked on Kan50 plate(s) and PCR’d. Master mix calcs are here. Cycling params:
Colony PCR - 5’RACE Colony: COX2 (repeat of yesterday’s PCR)
Repeated yesterday’s PCR on the re-streaked colony in order to run the product on a gel with a more appropriate ladder. See yesterday’s entry for all PCR info.
Colony PCR - 5’ RACE Colony: COX2
One white colony (marked with arrow in image linked) from the two plates from Steven’s cloning (from yesterday) was picked, restreaked on a new Kan50 plate (no X-gal) and PCR’d.
Colony PCR - 5’ RACE Colonies
Two light blue colonies (there were no white colonies) were picked, restreaked on a new Kan50 plate (no X-gal) and PCR’d.
Ligations - COX1/COX2 PCR Products
Performed ligations/cloning on a variety of COX1 genomic and COX 5’ RACE products using the TOPO TA Cloning Kit (Invitrogen). Used 2uL of gel-purified PCR product in each cloning rxn, 2.5uL of H2O, 1uL of salt solution, and 0.5uL of the Invitrogen pCR2.1 vector. Incubated samples for 5mins at RT. Used 2uL of the cloning reaction to transform TOP10 chemically competent cells (Invitrogen), mixed very gently, incubated on ice for 5mins, heat shocked at 42C for 30s and immediately placed on ice. Added 250uL of SOC Medium and incubated tubes at 37C, 200RPM for 1hr. Plated 50uL of each transformation on LB+Kan plates (made by Steven on unknown date with unknown Kan concentration) containing 40uL of 40mg/mL X-gal. Incubated O/N at 37C. Remaining volume of transformed bacteria were stored @ 4C.
5’/3’ RACE PCRs - Nested PCRs for COX2 Sequence
Due to the failure of the primary PCR on both 5’ and 3’ RACE cDNA libraries (from 20080619) yesterday, will perform nested PCR using a nested GSP designed by Steven (CgPGSRACEsrNGSP1; SR ID:1209). The 5uL of PCR reactions that were set aside yesterday were diluted to 250uL with tricine-EDTA (supplied with the Clontech SMARTer RACE cDNA Amplification Kit) as instructed in the Clontech manual. The master mix and tube layouts were exactly the same as yesterday’s, but instead of using 2.5uL of RACE cDNA library as template, I used 5uL of the diluted PCR reaction. Additionally, Universal Nested Primers were used instead of the Universal Primer Mix (both supplied in the kit). Cycling parameters followed “Program 2” from the Clontech manual for 25 cycles.
5’/3’ RACE PCRs - COX2 Sequence on 5’ & 3’ RACE Libraries (from 20080619)
Ran PCRs on both the 5’ & 3’ RACE libraries created 20080619 with a new COX2 gene-specifc (GSP) primer designed by Steven (CgPGSRACEsrGSP1; SR ID: 1208). Although this primer was designed to obtain additional 5’ sequence, it was used with both 5’ and 3’ libraries as a precaution in case it accidentally designed on the wrong strand. PCR rxn was set up according to the Clontech SMARTer RACE cDNA Amplification Kit. Master mix calcs are here. PCR cycling followed “Program 1” from the Clontech manual for 25 cycles.
qPCR - C.gigas COX1/COX2 Tissue Distribution
Performed qPCR using pooled cDNA from 20110311. Pooled 2uL from each of the following samples groups: Dg 3hr C, Gill 1hr C, Gill 1hr E, Mantle 3hr C, and Muscle 3hr C. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). Primers sets run were:
Reverse Transcription - C.gigas DNased RNA (from 20110131) from V.vulnificus Exposure & Tissues (from 20110111)
Performed RT on DNased RNA using Promega MMLV RT and Oligo dT according to manufacturer’s protocol, using 1ug of DNased RNA. Due to large number of samples, cDNA was made in PCR plate. Plate layout and calcs are here. cDNA was diluted 4-fold (to 100uL total volume) based on qPCR done by Emma on 20110202.
SOLiD Sequencing Submission
Submitted the following 8 samples for SOLiD sequencing at HTGU:
mRNA Isolation - Pooled Black Abalone Dg RNA (from Abalone Dg Exp 1)
mRNA was isolated for SOLiD sequencing by HTGU. Made two pools of San Nick RNA (Control and Exposed) using equal amounts (5ug) of each individual sample. Individual samples used can be found here. mRNA was isolated using Ambion’s Micro PolyAPurist Kit according to protocol. Procedure was performed two times on each pool and then EtOH precipitated. Samples were resuspended in 10uL of The RNA Storage Solution provided in the kit and spec’d on the Roberts Lab ND1000. Samples were stored @ -80C in the “Next Gen Sequencing Libraries” box.
3’RACE - C.gigas 3’RACE for COX2
Used Cg_COX2_3’RACE_short (SR ID: 1197) & Cg_COX2_3’RACE_long (SR ID: 1196) and the Clonetech SMART RACE cDNA Amplification Kit (unknown acquisition date) to attempt to acquire more 3’ sequence of the C.gigas COX2 isoform. Used Gigas 3’RACE cDNA (from 20080610).
NanoDrop1000 Comparison - Roberts vs. Young Lab
A previous comparison was performed (see 20110209), but it was determined that the standard DNA being used to test the machines was old/degraded. Lisa ordered a new standard DNA dilutions series (Quant-iT dsDNA Kit; Invitrogen) and these DNAs were used. All DNAs were measured 5 times and were mixed by gently flicking between each measurement. A “blank” was measured between each different [DNA] and, if the reading was > + or - 1ng/uL, the machine was reblanked.
Data Analysis - Young Lab ABI 7300 Calibration Checks
All runs (3 runs were conducted) were created using a master mix containing C.gigas gDNA (either 50ng or 100ng), 1X Promega qPCR Master Mix, 0.2uM each of forward/reverse primers (18s; Roberts SR ID: 156, 157). The master mix was mixed well and 10uL were distributed in each well of ABI plates. Plates were sealed with ABI optical adhesive covers.
qPCR - Test Young Lab qPCR Calibration
This is a repeat of the two runs from yesterday, just to see if there is a correlation between the failed plates being the first of the day or not. Master mix calcs and cycling params are here (these calcs are from yesterday, but were used again for today).
NanoDrop1000 Comparison - Roberts vs. Young Lab
Due to an apparent reduction in assay sensitivity for the Hematodidium qPCR assay, we have decided to determine if the spec readings of the plasmid DNA being used for the standard curves are accurate. Used C.gigas gDNA and the lambda DNA Standard (100ng/uL) included in the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) that was marked as received 9/1/10. Tested both the Roberts Lab and Young Lab using these DNAs. At least 6 separate measurements were taken of each DNA on each machine. Samples were briefly mixed by flicking the tube 4-5 times prior to each measurement.
qPCR - Test Young Lab qPCR Calibration (Repeat)
This was repeated from earlier today due to the failure of the previous run, but had to use new gDNA since I ran out of the stock I had previously used. Master mix calcs and cycling params are here.