GSM4976828: H3_MLN_ChIP-Seq_rep3; Homo sapiens; ChIP-Seq
For spike-in experiments (ChIP-Rx) 10% of fixed NIH-3T3 mouse cell lines were added before lysis. Cells were treated with 1% formaldehyde for 5 min at RT following 5 min of incubation with glycine. After cell lysis (5 mM PIPES pH 8.8, 5 mM KCl, 0.5% NP40), nuclei were resuspended in RIPA buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1% Triton-X-100, 0.1% deoxycholic acid (DOC), 0.1% SDS, 1 mM EDTA containing protease and phosphatase inhibitor cocktail) and DNA was fragmented to a size <500 bp using a Covaris M220 sonifier. Antibodies were bound to Protein A/G-Dynabeads® (Invitrogen) and immunoprecipitated. After extensive washing, chromatin was eluted with 1% SDS and crosslinking was reverted overnight. Chloroform/phenol extraction was used for DNA purification. Purified DNA was end-repaired, A-tailed, ligated to Illumina adaptors, size-selected (200 bp) and purified with a gel extraction kit. DNA fragments were amplified by 16 cycles of PCR and library size and amount of library was specified with the Fragment Analyzer (Agilent). The library was subjected to Illumina NextSeq 500 sequencing according to the manufacturer's instructions. After base calling with Illumina's FASTQ Generation software v1.0.0 (NextSeq 500 sequencing), high quality PF-clusters were selected for further analyses. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 16 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.