GSM9092814: ICM_Nsd1KO_WGBS_rep6; Mus musculus; Bisulfite-Seq
Cells were lysed in STEM-seq lysis buffer and subject to bisulfite conversion and subsequent library preparation. ICMs were lysed with 10 μl of STEM-seq lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, pH 8.0, 0.5% NP-40) containing 1 μl of protease K (NEB, P8107S) for 3 h at 55 °C, followed by heat-inactivation for 1 h at 72 °C. Samples were then treated with 1 μl of dsDNA Fragmentase (NEB; M0348AA) for 30 min before being subjected to bisulfite conversion using EZ DNA Methylation-Lightning Kit (ZYMO, D5030). Libraries were prepared using the EpiArt DNA Methylation Library Kit (Vazyme, NE103) according to the manufacturer's instructions. In brief, a 3' adaptor was first added to the converted and denatured ssDNA. After DNA extension and purification, a 5' adaptor was added. dsDNA with both 3' and 5' adaptors were then subjected to PCR amplification and libraries were sequenced using DNBSEQ-T7. For all samples, λ-DNA (Promega, D150A) was added with a mass ratio of 1:200 before bisulfite conversion for conversion rate analysis.