GSM1536372: NBM_5_hmeSeal_input; Homo sapiens; OTHER
DNA was extracted using Qiagen Puregene kit protocol; hMe-seal 5hmC labelling: Genomic DNA (gDNA) was sonicated into 200-400bp long fragments (set up: high, 30 times, 30s interval pulse followed by 30 s pause).  Subsequently 2 g units are wrong of gDNA were labeled with chemically modified uridine diphosphoglucose glucose (UDP-6-N3-Glu) by viral β-glucosyltransferase (β-GT) as described previously(Song et al., 2011, Nature Biotech.). Afterwards the labeled DNA was purified by QIAquick PCR Purification Kit (Qiagen Inc.,Valencia CA) and eluted in water.  The click chemistry reaction was performed by addition of 150 μM dibenzocyclooctyne modified biotin into the eluted DNA, the reaction mixture was incubated for 2 h at 37 °C.  The DNA samples were then purified by MinElute Reaction Cleanup Kit (Qiagen Inc., Valencia CA) and the amount of eluted DNA was determined by Nanodrop UV spectroscope (Thermo, Wilmington, DE) ERRBS:  Library construction protocol: Genomic DNA was purified from cell lines and primary human samples, digested with MspI restriction enzyme (200U MspI per reaction), subjected to end repair and adenylation followed by ligation to cytosine methylated paired-end Illumina adaptors (2000U T4 DNA ligase per reaction). Fragments of 150-250 bp (reduced representation bisulfite sequencing) and 150-400 bp (for enhanced reduced representation bisulfite sequencing) were isolated after agarose gel electrophoresis and subjected to bisulfite conversion (single conversion round using Zymo Research EZ DNA Methylation Kit) and PCR amplification using paired-end Illumina primers (18 cycles).  Libraries were sequenced using an Illumina Genome Analyzer II or HiSeq2000 per manufacturer’s recommended protocol for 50bp single end read runs. Bisulfite-seq (RRBS) and OTHER (hMe-Seal: chemical labeling,biotnylation and pulldown of regions with 5hmC )
GEO Accession
GSM1536372