GSM5058943: DNMT3A1-W330R_RRBS_parental; Mus musculus; Bisulfite-Seq
DNA was extracted from flash frozen cell pelets of about 2 million cells using DNeasy Blood & Tissue Kit (Qiagen, 69509) 100 ng of genomic DNA were digested with 100U of MspI (NEB) and end-repaired/A-tailed using Kapa Hyper Prep kit (Kapa Biosystems). After ligation of Illumina-sequencing compatible indexes, DNA was purified using a 1X Agencourt AMPure XP bead clean up (Beckman Coulter). Bisulfite conversion was carried out using the Zymo EZ DNA kit (Zymo Research) using the following program: 55 cycles: 95°C 30s, 50°C 15 min, 4°C hold. Libraries were amplified 17 cycles using Uracil+ Ready mix (Kapa Biosystems, KK2801). The resulting libraries, mean size 388bp, were normalized to 3nM, pooled and clustered on a pair end read flow cell and sequenced for 100 cycles on a NovaSeq6000 (Illumina).