CMS-IP for 5hmC Profiling in Pro-B Cells

CMS-IP was performed similar to pervious described (Huang et al., 2012 (link); Pastor et al., 2011 (link)). Briefly, genomic DNA from in vitro cultured pro-B cells was isolated with PureLink Genomic DNA kit (Thermo Fisher) and were spiked-in with cl857 Sam7 λDNA (Promega,Madison, WI) and PCR-generated, hmC-containing puromycin-resistant gene at a ratio of 200:1 and 100,000:1, respectively. DNA was sheared with a Covaris E220 (Covaris), end-repaired, A-tailed, ligated with methylated Illumina adaptors (NEB, Ipswich, MA), and bisulfite-converted (MethylCode Bisulfite Conversion Kit, Thermo Fisher). Bisulfite-converted DNA was denatured and immunoprecipitated with anti-CMS serum. Immunoprecipitated DNA was PCR-amplified with barcoded primers (NEBNext Multiplex Oligos for Illumina, NEB) for 15 cycles with Kapa HiFi Uracil+ (Kapa Biosystems, Wilmington, MA). Resulting libraries were sequenced with a HiSeq2500 system for 50 bp paired-end reads (Illuminia, San Diego, CA). The sequence reads were mapped to mm9 with Bismap, and CMS-enriched genomic regions were identified using the ‘findPeaks' command in HOMER with the 'histone’ mode and default parameters (Heinz et al., 2010 (link)).