title: GSM8965173: A549 cells, RNAP2 ChIP, I+B, 1h, rep 1; Homo sapiens; ChIP-Seq
library_construction_protocol: Protein-DNA cross-linking was performed by adding methanol-free formaldehyde to 1% in the culture medium and incubating for 10 min at room temperature. Formaldehyde was then quenched at room temperature for 5 min with 125mM glycine prior to washing cells for 5 min with ice-cold PBS and scraping into ice-cold lysis buffer (5mM PIPES pH 8.0, 1mM EDTA, 85mM KCl, 5% glycerol, 0.5% NP-40) supplemented with protease inhibitor cocktail (PIC). Cell suspensions were incubated at 4°C for 3 h with continuous agitation. Nuclei were collected by centrifugation (600 g, 5 min, 4°C) and resuspended in ice-cold nuclei lysis buffer (NLB) (1xPBS containing 1mM EDTA, 5% glycerol, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with PIC. Samples were sonicated at 4°C using a Bioruptor set for 28 – 30 high-power bursts with 30 s on-off cycles. Lysates were cleared by centrifugation (maximum speed for 15 min at 4°C) and supernatants used for immunoprecipitation. Protein G magnetic Dynabeads were preincubated with primary antibody (2.5µl/sample) overnight at 4 °C in NLB supplemented with PIC and 5 mg/ml BSA. After washing twice with ice-cold NLB plus PIC, the Dynabeads were incubated with cleared lysates overnight at 4°C in NLB supplemented with PIC and BSA. Beads were given 4 washes with ice-cold NLB containing 500mM NaCl, followed by 4 washes with ice-cold LiCl buffer (20mM Tris at pH 8.0, 1 mM EDTA, 250mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate). Crosslinks were reversed by incubation in TE buffer (10mM Tris-HCl,1mM EDTA, pH 8.0) supplemented with 0.7% SDS and 0.2 mg/ml proteinase K for 3h at 55 °C, then 16 h at 65°C. DNA was purified with a ChIP DNA Clean & Concentrator kit. ChIP-seq libraries were prepared using the NEB Ultra II kit.