Reduced representation bisulfite sequencing of WWB-1203: whole blood sample
Genomic DNA was extracted and purified from 200 L of whole blood samples using Monarch Genomic DNA Purification Kits (New England Biolabs, United Kingdom). Samples of ~200 ng genomic DNA were digested with 20 units of MspI (C*CGG) and 10 units of AluI (AG*CT) (New England Biolabs, United Kingdom) at 37C overnight (~16 hours). Digested DNA was purified using 1.8 AMPureXP magnetic beads (Agencourt, United States), then end-prepped and dA-tailed using the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, United Kingdom). Methylated adaptors were ligated to the end-repaired and dA-tailed DNA using the NEBNext Ultra II Ligation Module (New England Biolabs, United Kingdom), followed by USER enzymatic treatment. Ligated products were purified and size-selected for fragments shorter than 450 bp using 0.6 AMPureXP. Size-selected fragments were bisulfite-converted using Methylamp One-Step DNA Modification Kit (Epigentek, United States) according to the manufacturers protocol. These sub-libraries were barcoded and enriched using NEBNext Q5U Master Mix (New England Biolabs, United Kingdom). Sub-libraries were purified using 1.8 AMPureXP and pooled to equimolarity for the final library at ~20 nM, which was then sequenced on an Illumina NovaSeq 6000 system with paired-end mode of 150 bp.