GSM3102890: KI481_Input_BJ5ta; Homo sapiens; ChIP-Seq
Cells in culture dishes were crosslinked in 1% formaldehyde for 15 min and the reaction was quenched by 125 mM glycine. Cross-linked cells were washed with LB1 (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100) and then with LB2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 10 mM Tris-HCl pH 8.0). For cell-cycle-stage specific ChIP, cross-linked cells were stained by DAPI (5 µg/mL) in LB1 for 5 min at RT, sorted based on the cellular DNA content using BD FACS Aria II or BD FACS Aria IIIu, and then washed with LB2. Chromatin was extracted by sonication in LB3-Triton (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8, 100 mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroyl sarcosine, 1% Triton). LB1, LB2, and LB3-Triton used during extraction were supplmented with 1x protease inhibitor cocktail (Calbiochem 539131) and 100 nM phosphatase inhibitor Nodularin (Enzo ALX-350-061).  One million cells were used in a single 200-µL ChIP reaction. Antibodies used in ChIP are: rabbit monoclonal anti-phospho-Ser22-LMNA antibody D2B2E (Cell Signaling 13448S, Lot # 1; 5 µL per IP); mouse monoclonal anti-unphospho-Ser22-LMNA antibody E1 (Santa Cruz Biotechnology sc-376248, Lot # H2812; 10 µL per IP); mouse monoclonal anti-acetyl-Lys27 histone H3 antibody (Wako MABI0309, Lot # 14007; 2 µL per IP); mouse monoclonal anti-trimethyl-Lys4 histone H3 antibody (Wako MABI14004, Lot # 14004; 2 µL per IP); mouse monoclonal anti-Ty1 antibody (Diagenode C15200054, Lot # 005; 1 µL per IP). Immunoprecipitated DNA was reverse-crosslinked and used to construct high-throughput sequencing libraries using NEBNext Ultra DNA Library Prep Kit (New England Biolabs, E7370).