title: GSM3032525: Sham_rg108_RRBS_2; Rattus norvegicus; Bisulfite-Seq
library_construction_protocol: DNA was extracted using Phenol:Chloroform, followed by ethanol precipitation, and dissolved in 150 mL TE buffer (10 mM Tris-Cl pH 8.0, 0.5 mM EDTA). RRBS-seq was performed as described (Gu et al., 2011). 50 ng of purified DNA was digested with MspI (Fast digest MspI, Thermo Scientific FD0544) for 30 min at 37°C followed by heat inactivation at 65°C for 5 min. Library preparation was performed using NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, E7370L) and ligated with methylated adapters for Illumina at a dilution of 1:10 (New England BioLabs, E7535L). The adapter ligated DNA was subjected to bisulfite conversion with EpiTect fast bisulfite conversion kit (Qiagen, 59824) using the following cycling conditions: 2 cycles of (95°C; 5min, 60°C; 10min, 95°C; 5min, 60°C; 10min) and hold at 20°C. Bisulfite converted DNA was PCR amplified for 14-16 cycles using 2.5 U of Pfu Turbo Cx Hotstart DNA polymerase (Agilent Technologies, 600410) and size selected for fragments between 200 bp to 500 bp with Ampure Xp magnetic beads (Agencourt, A63880). The purified DNA was subjected to single end sequencing using the Illumina Hiseq 2500.