title: GSM2722165: mre_NS_d30; Homo sapiens; MRE-Seq
library_construction_protocol: DNA was isolated from cell pellets via phenol/chloroform extraction. MRE-seq was performed as in Maunakea et al. 2010, with modifications as detailed below. Five parallel restriction enzyme digestions (HpaII, Bsh1236I, SsiI (AciI) and Hin6I (Fermentas), and HpyCH4IV (NEB)) were performed, each using 1 µg of DNA per digest for each of the samples. Five units of enzyme were initially incubated with DNA for 3 h and then an additional five units of enzyme were added to the digestion for a total of 6 h of digestion time. DNA was purified by phenol/chloroform/isoamyl alcohol extraction, followed by chloroform extraction using phase lock gels. Digested DNA from the different reactions was combined and precipitated with one-tenth volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol. The purified DNA was size selected and purified (50-300 bp) by gel electrophoresis and Qiagen MinElute extraction. Library construction was performed as per the Illumina Genomic DNA Sample Prep Kit protocol with the following modifications. During the end-repair reaction, T4 DNA polymerase and T4 PNK were excluded and 1 µl of 1:5 diluted Klenow DNA polymerase was used. For the adapter ligation reaction, 1 µl of 1:10 diluted PE adapter oligo mix was used. Ten microliters from the 30 µl of purified adapter ligated DNA was used for the PCR enrichment reaction with PCR PE Primers 1.0 and 2.0. PCR products were size selected and purified (170- 420 bp) by gel electrophoresis and Qiagen QIAquick extraction. DNA libraries were checked for quality by Nanodrop (Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent). Reads were aligned to hg19 using BWA and pre-processed using methylQA. MRE reads were normalized to account for differing enzyme efficiencies and methylation values were determined by counting reads with CpGs at fragment ends (Maunakea et al., 2010). Detailed library construction protocols for MRE-seq are publicly available at the NIH Roadmap Epigenomics project website (http://www.roadmapepigenomics.org/protocols/type/experimental/).