GSM1463834: WGBS_3w_male; Mus musculus; Bisulfite-Seq
DNA was isolated from snap-frozen mouse tissues or from frozen human samples (purchased from Cancer Center Tissue and Tumor Bank of UMass Medical School) and incubated in lysis buffer (25  mM Tris-HCl (pH  8), 2  mM EDTA, 0.2% SDS, 200  mM NaCl) supplemented with 300 μg/ml proteinase K (Roche) followed by phenol:chloroform extraction and ethanol precipitation. Unmethylated lamba phage DNA was spiked in for a final concentration of 0.5%. 1 μg of gDNA was sonicated using The Covaris S2 Sonicator (Covaris Inc, Woburn MA) to the desired size range of approximately 125 bp median size, with the majority of insert sizes falling within the range of 100-200bp as determined by Agilent Bioanalyzer examination and quantified using the Kapa Biosystems kit. End repair, A-tailing, and adapter ligation are carried out using the standard Illumina TruSeq protocol.  Adapters used were the methylated bar coded adapters included in the Illumina TruSeq DNA sample preparation kit.  Sodium bisulfite conversion of all DNA samples following adapter ligation was performed using the EZ DNA Methylation-Gold Kit or the EZ DNA Methylation-Lightning kit  (Zymo Research). 4-8 rounds of PCR were performed using the KAPA HiFi +Uracil DNA polymerase (Kapa Biosystems). AMPure XP purification (Agencourt) was used to retain fragments with insert sizes between 100-200bp, and this insert size range was verified post-sequencing.