GSM5126368: WT-Day5.CMSIP1; Mus musculus; OTHER
Genomic DNA was isolated using Qiagen DNeasy Blood and Tissue kit. RNA was isolated using Qiagen RNeasy mini kit. CMS-IP library construction: DNA was sheared using Covaris Adaptive Focused Acoustic to the center at 200 bp. DNA fragments were size selected and ligated with methylated adaptors and treated with sodium bisulfite (invitrogen). The extra adapters were removed by AmpurXP (Beckman). The DNA was then denatured for 10 minutes at 95 °C (0.4 M NaOH, 10 mM EDTA), neutralized by the addition of cold 2M ammonium acetate (pH 7.0) for 10 min on ice. Then denatured DNA fragments were incubated with 1 μl anti-CMS antiserum in 1x IP buffer (10 mM sodium phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100) for 2 h at 4 °C, and then precipitated with protein A/G beads. Precipitated DNA was eluted with proteinase K, purified with phenol chloroform, amplified and enriched by 10-12 cycles PCR using Kapa HiFi uracil polymerase. RNA-seq library construction is followed standard illumina library construction protocols. MethylC-seq: DNA was sheared using Covaris Adaptive Focused Acoustic to the center at 300 bp. DNA fragments were size selected and ligated with methylated adaptors and treated with sodium bisulfite (invitrogen). Libraries were enriched by 4-6 cycles PCR using Kapa HiFi uracil polymerase.