GSM9262133: HCT116, Ab: P-SF3B1 (T235), experiment2; Homo sapiens; ChIP-Seq
ChIP was performed using the following antibodies for each biological technical replicate: 30 ul of P-SF3B1 (T211 or T235) in two technical replicates that were pooled together to obtain one biological replicate. In brief, 20 µl of Dynabeads protein G (Thermo Fisher Scientific, 10009D) per immunoprecipitation were pre-blocked with 0.2 mg/ml BSA (Thermo Fisher Scientific, AM2616) for 4 hours, washed 3× with RIPA buffer, followed by incubation with the specific antibody at 4 °C for at least 4 hours. HCT116 cells (70 % confluency) were treated with 50 nM SY-351 for 2 h. Cells were cross-linked with 1 % formaldehyde for 10 min, and the reaction was quenched with glycine (final concentration 125 mM) for 5 min. Cells were washed 2× with ice-cold PBS, scraped and split to several tubes so that there was about 20 µl packed cell pellet per tube. Each 20 µl packed cell pellet was lysed in 650 µl of RIPA buffer and sonicated 20× 7 s (amplitude 0.85) using a 5/64 probe (QSonica Q55A). Supernatant (13000 g/4 °C/10 min) was pooled and pre-cleared by incubating with Dynabeads protein G (Thermo Fisher Scientific, 10009D) at 4 °C for 2‒4 hours. Pre-cleared extracts were split into two technical replicates (900 µl each) and incubated overnight with antibody (P-CDK11, SF3B1)-bound Dynabeads protein G. Beads were sequentially washed once with low-salt buffer (20 mM Tris-Cl, (pH 8), 150 mM NaCl, 2 mM EDTA, 1% Triton X 100, 0.1% SDS), once with high-salt buffer (20 mM Tris-Cl, (pH 8), 500 mM NaCl, 2 mM EDTA, 1% Triton X 100, 0.1% SDS), once with LiCl buffer (20 mM Tris-Cl, (pH 8), 250 mM LiCl, 2 mM EDTA, 1% NP 40, 1% sodium deoxycholate) and 2× with TE buffer (10 mM Tris Cl, (pH 8), 1 mM EDTA). Immunoprecipitated complexes were eluted 2× 15 min with 250 µl of elution buffer (1% SDS, 0.1 M NaHCO3). NaCl was added to final concentration of 0.2 M and samples were incubated at 65 °C for at least 4 hours to reverse cross-linking. Tris, pH 7.8, proteinase K (Sigma-Aldrich, P5568) and GlycoBlue (Thermo Fisher Scientific, AM9516) were added to final concentration of 30 mM, 0.04 mg/ml and 0.06 mg/ml, respectively, and incubated at 42 °C for 2 hours. After phenol–chloroform extraction (Sigma-Aldrich, P3803), immunoprecipitated DNA was dissolved in 17 µl of water and technical replicates were pooled. ChIP–seq libraries were generated using KAPA Biosystems Hyper Prep Kit (Roche, KK8502) and NEBNext Multiplex Oligos for Illumina (New England Biolabs, E7335S, E7500S).