title: GSM1643142: HeLa-BATF2 Flag; Homo sapiens; ChIP-Seq
library_construction_protocol: HeLa-BATF2 cells were collected, washed with PBS, and treated with 1% formaldehyde for cross-linking reactions. The incubation with glycine for was then performed to quench the excess cross-linking reactants. Samples were exposed to the cold lysis buffer supplemented with mixture protease inhibitors to allow the release of nuclei. The nuclei were collected and resuspended in the nuclear lysis buffer, and then subjected to sonication. Anti-Flag and anti-IgG antibodies of total 5 ug were added to the nucleic supernatant for overnight incubation at 4℃. The protein-DNA complex bound to protein G-agarose was released from protein G-agarose with ChIP elution buffer. To break the cross-linking, NaCl at a final concentration of 0.2 M was added to the supernatant for overnight incubation. Finally, the DNA constituent was isolated by phenol-chloroform extraction; and the concentration was determined using a NanoDrop 2000c nucleic acid spectrophotometer (Thermo Scientific, USA). The immunoprecipitated DNA was subjected to the library preparation and sequenced on an Illumina platform at Novogene Life Sciences Company, China. The libraries were generated using Illumina TruseqTM DNA Sample Preparation Kit (Illumina, San Diego, USA) following manufacturer’s standard protocol. Indexing codes were added to each sample for identification. The remained overhangs were converted into blunt ends by exonuclease/polymerase treatments. Adenylation of the 3’ ends of DNA fragments was performed and the access enzyme was subsequently removed. Standard Illumina PE adapter oligonucleotides were ligated to the prepared DNA sample for hybridization. To isolate the preferred DNA fragments, agrose electrophoresis was used (120 V, 40 min, 1.5% agarose gel) to separate adapter-ligated constructs derived fragments from 400 bp to 500 bp in size. Following the purification procedures using spin column (QIAGEN, Dusseldorf, Germany), the DNA fragments with ligated adapters at both ends were enriched with Illumina PCR Primer Cocktail by a 10-cycles PCR reaction. The products were retrieved using a AMPure XP system (Beckman Coulter, Beverly, USA) and quantified by Agilent high sensitivity DNA assay on an Agilent Bioanalyzer 2100 equipment. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using a designated TruSeq PE Cluster Kit v3-cBot-HS (Illumia, San Diego, USA). After cluster generation, the obtained library was sequenced with an Illumina Hiseq 2000 platform where 100 bp paired-end reads were generated.
attributes.tag: GEO Accession
attributes.value: GSM1643142