GSM5990283: TKO_Dnmt3a2_W326R_RRBS_Rep1; Mus musculus; Bisulfite-Seq
Genomic DNA was purified from MEF-depleted ESCs using the QIAamp DNA Blood Mini kit. 80 ng of each sample was subjected to overnight digestion with 150 units of MspI (New England Biolabs) at 37 °C. The digest was purified by extraction with 25:24:1 phenol:chloroform:isoamyl alcohol (Thermo Fisher Scientific) and ethanol precipitation, followed by end-repair (End-It DNA End-Repair kit, Lucigen), A-tailing (Klenow fragment, New England Biolabs), and ligation to barcoded adapters (xGen Methyl UDI-UMI Adapters, Integrated DNA Technologies, 3 µL of 1.5 µM per ligation) using concentrated T4 ligase (New England Biolabs) at 16 °C overnight. Adapter-ligated DNA was purified by 1× SPRI, size-selected by gel extraction, bisulfite-converted (EZ DNA Methylation kit, Zymo; bisulfite conversion using the following thermocycler program: 55 cycles of 95 ˚C for 30 s, 50 ˚C for 15 min), and amplified using EpiMark hot start Taq DNA polymerase (New England Biolabs) and 17 cycles of PCR (KAPA library amplification primers).