title: GSM5963875: WGBS_ES_RF_#2_Muscle; Bos taurus; Bisulfite-Seq
library_construction_protocol: Blood cells and tissues were lysed in lysis buffer (0.05 M Tris-HCl (pH 8.0), 0.1 M EDTA, and 0.5% (w/v) SDS) with proteinase K (Fisher BioReagents, BP1700) at 55°C for four hours (for blood cells) or overnight (for tissues). Genomic DNA was extracted with Phenol:Chloroform:Isoamyl Alcohol (SIGMA, P3803) following the manufacturer's instructions. The concentration of DNA was measured by using a NanoDrop® ND-1000 Spectrophotometer (Thermo Fisher Scientific) and DNA integrity was confirmed by electrophoresis on a 0.7% agarose gel. Genomic DNA samples were stored at -20°C. This WGBS was conducted by CD Genomics (Shirley, NY, USA). Information on library preparation and sequencing obtained from the company is as follows: For WGBS library preparation, 1 ug of genomic DNA was fragmented by sonication to a mean size of approximately 200-400 bp. Fragmented DNA was end-repaired, 5'-phosphorylated, 3'-dA-tailed and then ligated to methylated adapters. The methylated adapter-ligated DNAs were purified using 0.8× Agencourt AMPure XP magnetic beads and subjected to bisulfite conversion by ZYMO EZ DNA Methylation-Gold Kit (zymo). The converted DNAs were then amplified using 25 μl KAPA HiFi HotStart Uracil+ ReadyMix (2X) and 8-bp index primers with a final concentration of 1 μM each. The constructed WGBS libraries were then analyzed by Agilent 2100 Bioanalyzer and quantified by a Qubit fluorometer with Quant-iT dsDNA HS Assay Kit (Invitrogen), and finally sequenced on Illumina Hiseq X ten sequencer. 0.1-1% lambda DNA were added during the library preparation to monitor bisulfite conversion rate.