title: GSM1199578: rCGG_Cb_5hmC_1; Mus musculus; MeDIP-Seq
library_construction_protocol: Cerebella samples were homogenized on ice by 2 ml Dounce tissue grinder in lysis buffer (100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 0.2% SDS (vol/vol), 200 mM NaCl). Proteinase K was freshly added to the buffer, and samples were kept at 55°C overnight. 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 at the second day, mixed completely, and centrifuged at top speed on desktop centrifugation for 10 min. Supernatant was then mixed with an equal volume of isopropanol to precipitate DNA. DNA pellets were washed with 70% ethanol and eluted with 10 mM Tris-HCl (pH 8.0). Chemical labeling-based 5hmC enrichment was applied. Briefly, DNA was sonicated to 100-500 bp, and then mixed with 100-μl solution containing 50 mM HEPES buffer (pH 7.9), 25 mM MgCl2, 250 μM UDP-6-N3-Glu, and 2.25 μM wild-type β-glucosyltransferase. Reactions were incubated for 1 h at 37°C. DNA substrates were purified via Qiagen DNA purification kit. 150 μM dibenzocyclooctyne modified biotin was then added to the purified DNA, and the labeling reaction was performed for 2 h at 37°C. The biotin-labelled DNA was enriched by Streptavidin-coupled Dynabeads (Dynabeads® MyOne™ Streptavidin T1, Life Technologies) and purified by Qiagen DNA purification kit. 5hmC-captured libraries were generated by the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina according to the manufacturer's protocol. In short, 25 ng of input genomic DNA or 5hmC-captured DNA were used. DNA fragments between 150 and 300 bp were gel-purified after the adapter ligation step. An Agilent 2100 BioAnalyzer was used to quantify the amplified DNA. 20-pM diluted libraries were eventually used for sequencing. We performed 38-cycle single-end sequencing 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.
attributes.tag: GEO Accession
attributes.value: GSM1199578