Whole-genome bisulfite sequencing (WGBS) of acute myeloid leukemia sample with engineered correction of DNMT3A R882H mutation (DNMT3A WT/WT), 4 months post-correction
Genomic DNA was extracted from >100,000 cells (except sample 6607#4, >47,000 cells) using DNA/RNA microprep plus kit (Zymo) following manufacturer's protocol. The generation of dual-indexed libraries for WGBS followed a modified protocol based on the NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs), in accordance with the manufacturer's instructions. Library input was >100 ng (except sample 6607#4, >40 ng) genomic DNA, quantified by the Qubit BR assay (Invitrogen), and 1% unmethylated Lambda DNA (Promega) was added as a control for bisulfite conversion efficiency. Fragmentation of input gDNA to achieve an average insert size of 350 bp was done with a Covaris S220 Focused-ultrasonicator. Size selection was performed using SPRIselect beads, isolating fragments within the 300-400 bp range. Bisulfite conversion post-size selection was done with the EZ DNA Methylation-Lightning Kit (Zymo), following the manufacturer's guidelines. For amplification post-bisulfite conversion, Kapa HiFi Uracil+175 (Kapa Biosystems) polymerase was used under the following cycling conditions: 98°C for 45 s, followed by 9 cycles of 98°C for 15 s, 65°C for 30 s, and 72°C for 30 s, with a final extension step at 72°C for 1 min. WGBS libraries were sequenced on an Illumina NovaSeq 6000 instrument using an S4 flowcell and a 150 bp paired-end dual indexed run, with 5% PhiX library control (Illumina).