GSM9017300: WT 293T rep1; Homo sapiens; ChIP-Seq
Freshly harvested cells were cross-linked with 1% formaldehyde (ThermoFisher Scientific #28906) for 10 min at room temperature, after which the reactions were quenched for 5 min by the addition of glycine to the final concentration of 0.125 M. Fixed cells were washed twice with PBS, snap frozen and store at -80°C. Frozen cell pellets were sent to MSKCC Epigenetics Research Innovation Lab for processing. Cells were resuspended in SDS buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5% SDS, 1x protease inhibitor cocktail from Roche). The resulting nuclei were spun down, resuspended in the immunoprecipitation buffer at 1 mL per 0.5 million cells (SDS buffer and Triton Dilution buffer (100 mM NaCl, 100 mM TrisHCl pH 8.0, 5 mM EDTA, 5% Triton X-100) mixed in 2:1 ratio with the addition of 1x protease inhibitor cocktail (Millipore Sigma, #11836170001) and processed on a Covaris E220 Focused-ultrasonicator to achieve an average fragment length of 200-300 bps with the following parameters: PIP=140, Duty Factor=5, CBP/Burst per sec=200, Time = 20 min. Chromatin concentrations were estimated using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific #23227) according to the manufacturer's instructions. The immunoprecipitation reactions were set up in 500uL of the immunoprecipitation buffer in Protein LoBind tubes (Eppendorf, #22431081) and pre-cleared with 50uL of Protein G Dynabeads (ThermoFisher Scientific #10004D) for two hours at 4°C. After pre-clearing, the samples were transferred into new Protein LoBind tubes and incubated overnight at 4°C with 10uL of the H3K27ac antibody (CST, 8173. The next day, 50uL of BSA-blocked Protein G Dynabeads were added to the reactions and incubated for 2 hours at 4°C. The beads were then washed two times with low-salt washing buffer (150mM NaCl, 1% Triton X-100, 0.1% SDS, 2mM EDTA, 20mM TrisHCl pH8.0), two times with high-salt washing buffer (500mM NaCl, 1% Triton X-100, 0.1% SDS, 2mM EDTA, 20mM TrisHCl pH8.0), two times with LiCL wash buffer (250mM LiCl, 10mM TrisHCl pH8.0, 1mM EDTA, 1% Na-Deoxycholate, 1% IGEPAL CA-630) and one time with TE buffer (10mM TrisHCl pH8.0, 1mM EDTA). The samples were then reverse-crosslinked overnight in the elution buffer (1% SDS, 0.1 M NaHCO3) and purified using the ChIP DNA Clean & Concentrator kit (Zymo Research, #D5205) following the manufacturer's instructions. After quantification of the recovered DNA fragments, libraries were prepared using the ThruPLEX®DNA-Seq kit (R400676, Takara) following the manufacturer's instructions, purified with SPRIselect magnetic beads (B23318, Beckman Coulter), and quantified using a Qubit Flex fluorometer (ThermoFisher Scientific) and profiled with a TapeStation (Agilent). The libraries were sent to MSKCC Integrated Genomics Operation core facility for sequencing on an Illumina NovaSeq 6000 (aiming for 30-40 million 100bp paired-end reads per library).