title: GSM1430728: CD4 naïve CMS-Seq IP BR1; Mus musculus; OTHER
library_construction_protocol: Genomic DNA was prepared by lysing T cells, followed by treatment with RNase A (Qiagen) for 1 hour at 37 0C and then overnight treatment with proteinase K (Roche) at 550C with vigorous shaking . DNA was purified after sequential treatment with phenol, phenol/chlorophorm/isoamyl alcohol, chlorophorm/isoamyl alcohol (all obtained from Sigma) and then ethanol (Sigma) precipitation. DNA was resuspended in 10 mM Tris-Cl pH 8.0, 0.1mM EDTA .In order to  prepare  Cytosine 5-methylenesulphonate immunoprecipitation (CMS-IP) sequencing libraries  genomic DNA was fragmented to an average size of 165 bp using  Covaris S2 (Covaris, Woburn, MA, USA). Genomic DNA was spiked with cl857 Sam7 Lambda DNA (Promega, Madison, WI) at a ratio of 200:1. The fragmented DNA was end repaired, 3' adenylated, ligated to methylated Illumina adaptor oligonucleotides (Truseq), and bisulfite converted. Bisulfite conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) was followed by immunoprecipitation of genomic DNA fragments containing CMS with a specific antiserum to CMS. Briefly, bisulfite converted genomic DNA was  denatured for 10 minutes at 95°C (0.4 M NaOH, 10 mM EDTA), then neutralized by addition of cold 2 M Ammonium Acetate pH 7.0 and incubation on ice for 10 minutes. Subsequently, the DNA was 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 2h at  4°C , and then precipitated overnight at 4°C  with Protein A and G magnetic beads (Invitrogen). 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 using the uracil insensitive Kapa HiFi HotStart Uracil+ (Kapa Biosystems).