GSM8772841: AMO1 shIRF4, Dox 0h, RNAPII-pS2 ChIP, Rep 2; Homo sapiens; ChIP-Seq
AMO-1 cells (10 x 10^6/condition) were double crosslinked by 50mM DSG (disuccinimidyl glutarate, #C1104 - ProteoChem) for 30-minutes followed by 10-minutes of 1% formaldehyde. Formaldehyde was quenched by the addition of glycine. Nuclei were isolated with ChIP lysis buffer (1% Triton x-100, 0.1% SDS, 150 mM NaCl, 1mM EDTA, and 20 mM Tris, pH 8.0). Nuclei were sheared with Covaris sonicator using the following setup: Fill level – 10, Duty Cycle – 15, PIP – 350, Cycles/Burst – 200, Time – 8-minutes). Sheared chromatin was immunoprecipitated overnight with the following antibodies: RNAPII-pS2 (Abcam – ab5095), IRF4 (Cell Signaling – 4964S). Antibody chromatin complexes were pulled down with Protein A magnetic beads and washed once in IP wash buffer I. (1% Triton, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC), twice in IP wash buffer II. (1% Triton, 0.1% SDS, 500 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC), once in IP wash buffer III. (0.25 M LiCl, 0.5% NP-40, 1mM EDTA, 20 mM Tris, pH 8.0, 0.5% NaDOC) and once in TE buffer (10 mM EDTA and 200 mM Tris, pH 8.0). DNA was eluted from the beads by vigorous shaking for 20-minutes in elution buffer (100mM NaHCO3, 1% SDS). DNA was de-crosslinked overnight at 65C and purified with MinElute PCR purification kit (Qiagen). DNA was quantified by Qubit and 10 ng DNA was used for sequencing library construction with the Ovation Ultralow Library System V2 (Tecan) using 14 PCR cycles according to the manufacturer's recommendations.  Libraries were sequenced with Illumina Nextseq, using paired-end 50bp read configuration.