Constructed next generation libraries (Illumina) using the bisulfite-treated DNA from yesterday using the EpiNext Post-Bisulfite DNA Library Preparation Kit - Illumina (Epigentek). Samples were processed according to the manufacturer’s protocol up to Section 8 (Library Amplification) with the following changes:
- Skipped Section 7.1 (recommended to do so in the protocol due to low quantity of input DNA)
Samples were stored O/N @ -20C.
dA Tailing Master Mix
10x Tailing Buffer 1.5uL x 17.6 = 26.4uL
Klenow 1uL x 17.6 = 17.6uL
H2O 0.5uL x 17.6 = 8.8uL
Add 3uL of master mix to each sample
Adaptor Ligation
2x Ligation Buffer 17uL x 17.6 - 299.2uL
T4 DNA Ligase 1uL x 17.6uL = 17.6uL
Adaptors 1uL x 17.6 = 17.6uL
Added 19uL of master mix to each sample
dsDNA Conversion Master Mix
5x Conversion Buffer 4uL x 17.6 = 70.4uL
C.P. 2uL x 17.6 = 35.2uL
H2O 3uL x 18.6 = 52.8uL
Add 9uL of master mix to each sample
End Repair
10x Buffer 2uL x 17.6 = 35.2uL
Enzyme 1uL x 17.6 = 17.6uL
H2O 5uL x 17.6 = 88uL
Added 8uL of master mix to each sample