Name: GSM8335309
Instrument: Illumina NovaSeq 6000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Frozen cell pellets were resuspended in SDS buffer (100mM NaCl, 50mM Tris-HCl pH 8.0, 5mM EDTA, 0.5 % SDS, 1x protease inhibitor cocktail from Roche). The resulting nuclei were spun down, resuspended in the immunoprecipitation buffer at 1mL per 0.5 million cells (SDS buffer and Triton Dilution buffer [100mM NaCl, 100mM Tris-HCl pH 8.0, 5mM EDTA, 5% Triton X-100] mixed in 2:1 ratio with the addition of 1x protease inhibitor cocktail) and processed on a Covaris E220 Focused-ultrasonicator to achieve an average fragment length of 200-300bps with the following parameters: PIP=140, Duty Factor=5, CBP/Burst per sec=200, Time = 1200s. Chromatin concentrations were estimated using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific) according to the manufacturer's instructions. The immunoprecipitation reactions were set up in 500uL of the immunoprecipitation buffer in Protein LoBind tubes (Eppendorf) and pre-cleared with 50 µL of Protein G Dynabeads (ThermoFisher Scientific) 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 the indicated antibodies (Key Resources Table). The next day, 50 µL 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 (150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), two times with high-salt washing buffer (500 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), two times with LiCL wash buffer (250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 % Na-Deoxycholate, 1 % IGEPAL CA-630) and one time with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The samples were then reverse-crosslinked overnight in elution buffer (1 % SDS, 0.1M NaHCO3) and purified using the ChIP DNA Clean & Concentrator kit (Zymo Research) following manufacturer instructions. After quantification of the recovered DNA fragments, libraries were prepared using the ThruPLEX®DNA-Seq kit (Takara) following the manufacturer's instructions, purified with SPRIselect magnetic beads (Beckman Coulter), and quantified using a Qubit Flex fluorometer (ThermoFisher Scientific) and profiled with a TapeStation (Agilent).