alias: GSM2466467
title: GSM2466467: ZNF100-replicate 2; Homo sapiens; ChIP-Seq
library_construction_protocol: Chromatin was prepared by resuspending 3x107 293T cells in 6 mL of PBS, adding 375 µL of methanol-free formaldehyde (16% - 1% final) and incubating for 10 min on a rotating wheel, quenching with Tris pH 8.0 (250 mM final) for 10 min on a rotating wheel, rinsing with PBS twice and freezing the pellet at -80°C. Nuclear extraction was performed as follow: fixed cell pellets were thawed on ice and resuspended in 1.5 mL of LB1 buffer (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP40, 0.25% Tx100) and incubated for 10 min at 4°C on a rotating wheel. The mixture was centrifuged at 1,700g at 4°C, the supernatant removed, the pellet resuspended in 1 mL LB2 buffer (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and incubated for 10 min at 4°C on a rotating wheel. The same procedure was repeeated 2 more times, replacing LB2 with SDS shearing buffer (10 mM Tris pH 8.0, EDTA 1 mM, SDS 0.15%) and the pellet was finally resuspended in 1 mL of this last buffer. Every buffer was prechilled and contained a protease inhibitor cocktail (cOmplete ULTRA Tablets EDTA-free, Roche). The suspension was transferred to a milliTUBE 1 ml AFA Fiber (part #520130) and processed in batch of 24 on an E220 focused-ultrasonicator from Covaris Inc. (Woburn, Massachusetts, USA) with the following settings: 20 min at 5% duty cycle, 140W, 200 cycles. The lysate was transferred to a 1.5 mL Eppendorf, centrifuged at 10,000g for 5 min to clear insoluble material – the supernatant was either frozen at -80°C or used right away for chromatin immunoprecipitation. Quality control was performed on a Bioanalyzer 2100 (Agilent) to verify that most fragments were between 200 and 600 bp. Chromatin immunoprecipitation was performed using anti-HA.11 antibody (clone 16B12, Covance), 15 µg per IP, and Protein G Dynabeads (ThermoFisher). ChIP-exo was performed on bead-bound chromatin as described (Rhee and Pugh, 2011), with adaptations for use in 96-well plates and Illumina sequencing (detailed protocol available in Supplemental Information of the manuscript associated with this GEO entry).
identifiers.namespace: GEO
identifiers.id: GSM2466467