title: GSM1173798: CMS-IP_mm_fc_10wk_IP; Mus musculus; OTHER
library_construction_protocol: Genomic DNA was purified from cells using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Cytosine 5-methylenesulphonate immunoprecipitation (CMS-IP) sequencing libraries were made as follows. Genomic DNA was fragmented to 100-200 bp using a Covaris S2 (Covaris, Woburn, MA, USA), end repaired, 3' adenylated, ligated to methylated Illumina adaptor oligonucleotides, and bisulfite converted as per the MethylC-Seq protocol, descibed previously [Lister R. et al., Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471, 68-73 (2011)]. Bisulfite conversion of hmC to cytosine 5-methylenesulphonate (CMS) was followed by immunoprecipitation of genomic DNA fragments containing CMS with a specific antiserum to CMS. Bisulfite converted genomic DNA was first denatured for 10 minutes at 95°C (0.4 M NaOH, 10 mM EDTA), neutralized by addition of cold 2 M Ammonium Acetate pH 7.0, incubated with 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 to overnight at 4°C, and then precipitated with Protein G beads. Precipitated DNA was washed 3 times with 1x IP buffer and eluted with Proteinase K, then purified with Phenol Chloroform. Following immunoprecipitation, sequencing libraries were amplified by 8 cycles of PCR.