GSM5769211: HMLE KO MaSC ChIP MED1 rep2; Homo sapiens; ChIP-Seq
ChIP-Seq for MED1 was performed as previously described (Holoch et al., 2021) with minor modifications. Briefly, WT and KO MaSC HMLE were FACS-sorted and expanded in culture for 72 h before being cross-linked in the culture dishes by incubation at RT in pre-warmed (37˚C) cross-linking medium (DMEM supplemented with 15 mM HEPES-NaOH pH 7.9, 15 mM NaCl, 0.15 mM EDTA, 0.075 mM EGTA and 1% formaldehyde). After 10 min, the cross-linking reaction was stopped by addition of 0.125 M glycine at RT for 5 min. Cells were then rinsed with cold PBS, scraped from the dishes, and transferred to 50-mL conical tubes, pelleted, and washed again in PBS. Next, cells were lysed by incubation at 4°C for 10 min in 1 mL buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 supplemented with protease inhibitors). Nuclei were pelleted, resuspended in 1 mL of buffer 2 (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA plus protease inhibitors) and rocked at RT for 10 min. Nuclei were pelleted again, resuspended in 1.3 mL of buffer 3 (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 0.5% N-lauroyl-sarcosine supplemented with protease inhibitors) and sonicated using a pre-cooled Diagenode Bioruptor for 30 min (30 s ON/30 s OFF). Solubilized chromatin was recovered by centrifuging at 20,000xg and saving the supernatant. To determine chromatin concentration, 20 μL of chromatin extract were de-crosslinked o/n at 65˚C in 180 μL of T50E10S1 (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS) and were purified the next day by phenol:chloroform extraction and ethanol precipitation. 10 μg of chromatin per sample were used to immunoprecipitate MED1. Sample volumes were equalized using buffer 3. For each sample, 50 µL of Protein A Dynabeads slurry were washed in 500 µL 0.5% BSA in PBS (3X) and then rocked at 4˚C for several hours in 200 µL 0.5% BSA in PBS and 10 µg of MED1 antibody. Just prior to the IP, 0.5 volume buffer 4 (15 mM EDTA, 3% Triton X-100, 0.3% sodium deoxycholate with protease inhibitors) was added to each chromatin sample, and 1/10th of the resulting volume was set aside as input. For each sample, Dynabeads were washed in 500 µL 0.5% BSA in PBS (3X), resuspended in 50 µL of a 2:1 mixture of buffers 3 and 4, and added to the chromatin extract. After an o/n IP at 4˚C (on a rotating wheel), beads were washed 6X in 0.4 mL ice-cold buffer 5 (50 mM HEPES-KOH pH 7.5, 0.5 M LiCl, 10 mM EDTA, 0.7% sodium deoxycholate, 1% NP-40 with protease inhibitors), with 2 min RT rotation per wash, and 1X in 0.4 mL 10 mM Tris-HCl pH 8, 50 mM NaCl, 1 mM EDTA. Precipitated chromatin was then eluted by incubating beads in 200 µL T50E10S1 at 65°C for 30 min (with vigorous shaking). The IP chromatin samples and the input were incubated o/n at 65°C to reverse cross-links, and then treated with 80 μg of Rnase A at 37°C for 1 h and 40 μg of proteinase K at 55°C for 1 h. DNA was extracted with phenol:chloroform and precipitated in ethanol. ChIP-Seq libraries were prepared using the Accel-NGS 2S Plus DNA library kit and sequenced on an Illumina Novaseq 6000 (PE100) at the Institut Curie Next Generation Sequencing Core Facility.