Bisulfite NGS Library Prep - Bisulfite Conversion & Illumina Library Construction of C.gigas larvae DNA

Bisulfite Conversion

The previous attempt at constructing a library for the 1000ppm larvae samples failed. I had previously sheared, quantified, and concentrated the DNA from this sample. As I had done previously, I combined 50ng from each of the two 1000ppm samples for a total of 100ng, and brought the sample volume up to 24μL with NanoPure H2O.

Bisulfite conversion was performed with the Methylamp DNA Modification Kit (Epigentek) according to the manufacturer’s protocol.

Sample was eluted with 10μL of Buffer R6 for immediate use.

Library Prep

Bisulfite Illumina library was made with [EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek)(https://github.com/sr320/LabDocs/blob/master/protocols/Commercial_Protocols/Epigentek_PostBisulfiteIlluminaLibraryPrep_P-1055.pdf).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.

  • Skipped Step 7.1 (per manufacturer’s recommendation for samples starting with <200ng)

  • Ran 13 cycles during the library amplification step (per manufacturer’s recommendation for samples starting with 100ng)

Sample was transferred to 1.5mL snap cap tube and stored @ -20C.  Will quantify library on Monday when Jake is also finished with his 12 libraries.