title: GSM3768260: ChIPseq_Histones_PC3_input; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP experiments were performed as previously described (Leone et al., 2017). Briefly, 1% formaldehyde was added to cultured cells to cross-link proteins to DNA. Isolated nuclei were then lysed (50 mM Tris-HCl pH 8.1, 10 mM EDTA and 1% SDS) and sonicated using a Bioruptor ultrasonic cell disruptor (Diagenode) to shear genomic DNA to an average fragment size of 200 bp. 20 μg of chromatin was diluted tenfold with ChIP buffer (16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS and 1.1% Triton X-100) and precleared for 2h with 10ul of packed Sepharose beads for at least 2h at 4°C. Immunoprecipitation was done overnight with the indicated antibodies. Pulldowns were done with Dynabeads protein-A (or -G, Millipore) for 4h at 4°C. For HA-TIP5 ChIPs, preclearing and pulldowns were done with beads block with BSA (50μg) and ytRNA (50μg), per 400ul of Dynabeads protein-A. After washing, elution and reversion of cross-links, eluates were treated with RNAse A (1μg). DNA was purified with phenol-chloroform, ethanol precipitated and quantified by quantitative PCR. The quantity and quality of the isolated DNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The NEBNext® Ultra™ II DNA Library Prep Kit from NEB (New England Biolabs, Ipswich, MA) was used in the succeeding steps. ChIP samples (1ng) were end-repaired and adenylated.  Adapters containing the index for multiplexing were ligated to the fragmented DNA samples. Fragments containing adapters on both ends were enriched by PCR.  The quality and quantity of the enriched libraries were measured using Qubit® (1.0) Fluorometer and the Tapestation (Agilent, Waldbronn, Germany). The product is a smear with an average fragment size of approximately 300 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 2500 ot Hiseq 4000 single end 125 bp using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA).
attributes.tag: GEO Accession
attributes.value: GSM3768260