alias: GSM1871924
title: GSM1871924: 1196; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA was isolated from brain samples with standard protocols. Tissues were homogenized on ice and then treated with proteinase K (0.667 µg/µl) in digestion buffer (100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 0.2% SDS (vol/vol), 200 mM NaCl) overnight at 55 °C. On the second day, an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, saturated with 10 mM Tris (pH 8.0) and 1 mM EDTA; P-3803, Sigma) was added, mixed completely, centrifuged for 5 min at 20,817xg, and precipitated with an equal volume of isopropanol. Genomic DNA was recovered in 10 mM Tris-HCl (pH 8.0) and sonicated to ~500 bp by Misonix 3000 (microtip, 4 pulses of 27 s each, 1-min rest on ice, output 2). 5-hmC enrichment was performed as previously described [18]. 5-hmC labeling reactions were performed in a 100 µl solution containing 50 mM HEPES buffer (pH 7.9), 25 mM MgCl2, 300 ng/µl sonicated genomic DNA (100-500 bp), 250 µM UDP-6-N3-Glu, and 2.25 µM wild-type _-glucosyltransferase. Reactions were incubated for 1 h at 37°C. DNA substrates were purified via Qiagen DNA purification kit or by phenol-chloroform precipitation and reconstituted in H2O. Click chemistry was performed by adding of 150 µM dibenzocyclooctyne-modified biotin into the DNA solution and incubating for 2 h at 37°C. Samples were purified by Pierce Monomeric Avidin Kit (Thermo) following the manufacturer's recommendations. After elution, biotin-5-N3-gmC-containing DNA was concentrated with 10 K Amicon Ultra 0.5-ml Centrifugal Filters (Millipore) and purified using a Qiagen DNA purification kit. Libraries were generated following the Illumina protocol for “Preparing Samples for ChIP Sequencing of DNA” (Part# 111257047 Rev. A). We used 25 ng of input genomic DNA or 5-hmC–captured DNA to initiate the protocol. DNA fragments of ~150–300 bp were gel-purified after the adaptor ligation step. PCR-amplified DNA libraries were quantified on an Agilent 2100 Bioanalyzer and diluted to 6–8 pM for cluster generation and sequencing. We performed 38-cycle single-end sequencing Generation using Version 4 Cluster Generation and Sequencing Kits (Part #15002739 and #15005236 respectively) and Version 7.0 recipes. Image processing and sequence extraction were done using the standard Illumina Pipeline.
identifiers.namespace: GEO
identifiers.id: GSM1871924