title: GSM7818549: DNMT3B_KO_DNMT3B_W263A_exp_2_WGBS; Homo sapiens; Bisulfite-Seq
library_construction_protocol: To extract genomic DNA cells were resuspended in genomic lysis buffer (300 mM NaCl, 1% SDS, 20 mM EDTA) and incubated with Proteinase K (Roche) at 55 C overnight. RNA was removed by incubation with RNase A/T1 Cocktail (Ambion) at 37°C for 1 h, in between two phenol-chloroform extraction steps. DNA was quantified by Nanodrop 8000 spectrophotometer and purity assessed by electrophoresis. 100 or 200 ng of purified DNA samples were sheared using the Covaris E220 Evolution Focused Ultrasonicator to create fragments of approximately 350 bp. 0.5 ng of unmethylated phage-λ DNA (NEB) was spiked into each DNA sample prior to shearing to allow assessment of the efficiency of the bisulfite-conversion reaction. 100 ng of each sheared DNA sample was then processed using the EZ DNA Methylation Gold Kit or EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer's protocol to create bisulfite-converted single-stranded (BC-ss) DNA. For WGBS experiment 1, the TruSeq DNA Methylation Kit (Illumina Inc.) was used.  Bisfulfite converted single-stranded DNA was randomly primed with a polymerase able to read uracil nucleotides to synthesise DNA strands containing a sequence specific tag. 3' ends of the newly synthesized DNA strands were then selectively tagged with a second specific sequence tag to produce di-tagged DNA molecules with known sequence tags at 5' and 3' ends. Di-tagged molecules were then enriched by PCR with unique indexed PCR primers to provide dsDNA libraries with Illumina sequencing adapters that could be multiplexed and sequenced on a single flow cell. For all other WGBS experiments the Accel-NGS Methyl-Seq DNA Library Kit (Swift BioSciences) was used. For the Swift kit, bisfulfite converted single-stranded DNA was first denatured before simultaneous tailing and ligation of truncated adapters to 3' ends with Adaptase. An extension step then incorporates the truncated adapter 1 by a primer extension reaction, before a ligation step adds truncated adapter 2 to the bottom strand only. 9 cycles of PCR were used to increase yield and incorporate full-length adapters for unique dual-indexed sequencing. Finally, Agencourt AMPure XP beads (Beckman Coulter) were used to remove oligonucleotides and small fragments and change enzymatic buffer composition. Libraries were quantified using the Qubit dsDNA HS assay kit, assessed for size and quality using the Agilent Bioanalyser with the DNA HS Kit and combined in a single equimolar pool. Sequencing was performed on a P2 flow cell on the Illumina NextSeq 2000 platform using NextSeq 1000/2000 P2 Reagents v3 kit (200 Cycles), or on the NextSeq 550 platform using NextSeq 500/550 High-Output v2 kit (150 cycles). PhiX Control v3 Library was spiked in at a concentration of ~5% to increase library diversity for sequencing. Library preparation and sequencing was performed by the Edinburgh Clinical Research Facility.