GSM8728093: A1-SUZ12-ChIP, mESC; Mus musculus; ChIP-Seq
Cross-linking ChIP in mESCs was done using ~2 × 107 cells per immunoprecipitation. Before fixation, medium was aspirated, cells were collected by TrypLE Express Enzyme (Gibco), and cells washed once with PBS. Cells were cross-linked (1% paraformaldehyde for 5 min at room temperature (RTo)) with gentle shaking. Glycine was added to quench (final concentration 125 mM, incubated for 5 min at RTo), then cells washed once with cold PBS and pelleted. To obtain a soluble chromatin extract, cells were resuspended in 1 ml LB1 (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 and 1× Complete protease inhibitor) and incubated rotating at 4 °C for 10 min. Samples were centrifuged, resuspended in 1 ml LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 1× Compete protease inhibitor), and incubated rotating at 4 °C for 10 min. Finally, samples were centrifuged, resuspended in 1 ml LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine, 1% Triton X-100 and 1× Complete protease inhibitor) and homogenized by passing two times through a 27-gauge needle. Chromatin extracts were sonicated for 14 min using a Covaris E220 focused ultrasonicator at peak power 140, duty factor 5, and cycles/burst 200. For ChIP-Rx of histone post-translational modifications, after centrifugation, samples were spiked with soluble chromatin from Drosophila S2 cells to comprise 2–5% of total chromatin in the lysate. ChIP and CUT&RUN libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to the manufacturer's instructions. All libraries were analyzed by TapeStation prior to sequencing. Single-end sequencing was performed on the Illumina NextSeq 500 sequencer.