title: GSM6047419: Batch_07_ChIPseq_TI; 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). Total inputs and chromatin immunoprecipitated samples were prepared for libraries using 10 ng of material. After end-repair and A-tailing, Illumina IDT indexes were ligated to the samples. Aliquots were tested in qPCR to determine the optimal number of PCR cycles needed to amplify each library without reaching saturation. Libraries were amplified with, size-selected using Ampure XP beads (Beckman Coulter), and always quality-checked on a Bioanalyzer DNA high sensitivity chip (Agilent) and quantified with a Qubit dsDNA HS assay kit (Qubit 2.0 Fluorometer, Invitrogen)