GSM4395119: TSB23_BJ_Young_anti-p65; Homo sapiens; ChIP-Seq
Fixed chromatin immunoprecipitation assays were performed as previously described (Li et al., 2013), with the following modifications: Samples prepared for ChIP of histone modifications were fixed in 1% formaldehyde for 10 minutes at room temperature, while samples prepared for ChIP of transcription factors were double cross-linked in 2mM DSG alone for 45 minutes at room temperature followed by 10 minute fixation with 1% formaldehyde.  After neutralization with 0.125M glycine, samples underwent nuclear extraction, and were afterwards resuspended in nuclear lysis buffer (50mM Tris/HCl pH7.4, 1% SDS, 10mM EDTA, 1x dissolved cOmplete Protease Inhibitor Cocktail (Sigma 11836145001)), and samples were sonicated using the Bioruptor (Diagenode).  The concentration of chromatin in each sample was detected using the Qubit Fluorometer (Thermo Fisher), and samples were then diluted 1:10 in dilution buffer (20mM Tris/HCl pH7.4, 100mM NaCl, 0.5% Triton-X-100, 2mM EDTA, 1x dissolved cOmplete Protease Inhibitor Cocktail).  Between 20-50 ug of chromatin from each sample was then used for each histone or transcription factor ChIP assay, and the volume of each sample was raised to 0.5 mL using 1:10 sonication:dilution buffer mixture.  Antibodies were then incubated with each sample overnight.    Antibody:chromatin complexes were then immunoprecipitated as described using 20 uL Protein G Dynabeads (Thermo Fisher), which were then washed using the following washes:  ChIPs of histone modifications were washed with the following buffers in order: 2xWB1 (20mM Tris/HCl pH7.4, 150mM NaCl, 1% Triton-X-100, 0.1% SDS, 2mM EDTA), 1xWB2 (20mM Tris/HCl pH7.4, 500mM NaCl, 1% Triton-X-100, 2mM EDTA), 1xWB3 (10mM Tris/HCl pH7.4, 250mM LiCl, 1% NP-40, 1% Deoxycholate, 1mM EDTA), 2xTE (10mM Tris/HCl pH 8.0, 1mM EDTA).  ChIPs of transcription factors were washed with the following buffers in order: 2xWB1 (20mM Tris/HCl pH7.4, 150mM NaCl, 1% Triton-X-100, 2mM EDTA), 1xWB2 (20mM Tris/HCl pH7.4, 300mM NaCl, 1% Triton-X-100, 2mM EDTA), 1xWB3 (20mM Tris/HCl pH7.4, 400mM NaCl, 1% Triton-X-100, 2mM EDTA), 2xTE (10mM Tris/HCl pH 8.0, 1mM EDTA).  After washing, samples were eluted overnight at 65 °C as described, purified using the QIAquick PCR Purification Kit (QIAGEN), and were eluted in EB (1% SDS, 0.1M NaHCO3).  Samples were then further sonicated for 10 minutes to ensure the majority of purified chromatin fell between 100-300bp. Purified chromatin was then prepared for deep sequencing with the Illumina HiSeq 2500 system using either the KAPA LTP Library Preparation Kit (KK8232) or KAPA Hyper Prep Kit (KK8500) for histone or transcription factor ChIPs, respectively, following manufacturer's instructions.  Samples were run on the Illumina HiSeq 2500 as single-read reactions of 50 cycles.