GSM9571579: hsc_w326r_s4; Mus musculus; OTHER
Genomic DNA was extracted using QIAamp UCP DNA micro kit (QIAGEN). Libraries were prepared from 0.5-1 ng of each sample using the NEBNext Enzymatic Methyl-seq Kit (NEB #E7120) according to manufacturer instructions for low input samples. Briefly, DNA controls were spiked in: unmethylated lambda DNA and CpG-methylated pUC19 DNA. Controls were diluted 1:500 and 1 mL of the relevant diluted controls were added to each DNA sample. Then, DNA samples were sheared to an average size of 240-290bp using Covaris E220 Evolution Focused Ultrasonicator (Covaris #3814). Sheared DNA samples were end-repaired, 5' phosphorylated and 3' dA-tailed. After adapter ligation, DNA fragments were purified with NEBNext Sample Purification Beads followed by Enzymatic Methyl-seq two-step conversion. Fragments were then purified with AMPure XP beads (1X volume) before PCR with unique dual index sequences to selectively enrich DNA fragments with adapter sequences and allow multiplex sequencing. Libraries were quantified by Qubit. Sequencing was performed on NextSeq 2000 (Illumina, #20038897) using P3 Reagents (#20040560).