GSM5267254: ChIP_Input_WT_rep1; Homo sapiens; ChIP-Seq
[isolation protocol]  Chromatin immunoprecipitation (ChIP) was performed on 3-5x10^6 cells per replicate using the ChIP Assay Kit (Cat. No. 17-295, Millipore).  All stages beyond the fixation stage used kit solutions as per the manufacturer's instructions.  A single 10 minute 1% formaldehyde fixation was used for all antibodies.  The cross-linking reaction was quenched by addition of glycine (Sigma-Aldrich) to a final concentration of 130mM), then washed once in PBS and snap frozen.  Samples were stored at -80C prior to further processing. Cells were lysed in SDS lysis buffer with Complete Proteinase Inhibitor Cocktail (PIC, Roche).  Samples were then fragmented using the Covaris S220 Focused ultra-sonicator with the following settings (Duty Cycle: 2%, Intensity: 3.0, Cycles/burst: 200, Power mode: Freq. Sweeping, Duration: 120s, Temp: 6C, Batches: 4).  Sonicated chromatin samples were diluted 1:10 in ChIP dilution buffer containing PIC to 2mL.  Before addition of the antibody, 100 μL was taken from each 2mL sample to be used as a 5% input.  The chromatin was pre-cleared with Protein A agarose for 30 min before immunoprecipitation overnight at 4C rotating (the volumes added are also shown).  Protein A agarose beads were added the next morning for 60 min at 4C, after which the beads were sequentially washed in Low Salt Buffer (1x), High Salt Buffer (1x), LiCl Buffer (1x) and TE Buffer (2x) with the final TE buffer wash at room temperature.  Bound chromatin fragments were eluted from beads in elution buffer (1% SDS, 0.1M NaHCO3), de-crosslinked by incubating samples at 65C for 5 hours, then proteinase K treated for one hour at 55C.  DNA was precipitated using phenol/chloroform extraction and ethanol precipitation with GlycoBlue (15mg/mL) (Thermo Scientific) to visualise the pellet prior to dissolving in 55 μL of H2O. DNA was purified by phenol-chloroform extraction with PhaseLock tubes (5Prime) and ethanol precipitation with NaOAc, and 2 µl GlycoBlue (Invitrogen). ChIP enrichment was determined by RT-qPCR NEBNext Ultra II DNA Library Prep kit for Illumina (New England Biolabs).