GSM2070451: NDTiF; Homo sapiens; ChIP-Seq
Chromatin used in ChIP: Chromatin was prepared from 107 cells as followed: adherent stem cells were detached with TrypLE, diluted in DMEM KO medium and washed with PBS. Cells were fixed with methanol-free formaldehyde (333 mM final) 10min at room temperature, diluted with Tris pH8 (200 mM final), washed 2 times with cold PBS and pellets were frozen at -80°C. Cross-linked cells were incubated 10min at 4°C in buffer I (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, supplemented with protease inhibitors), spinned, incubated 10min at 4°C in buffer II (10 mM Tris pH8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, proteases inhibitors), spinned, washed 3 times at 4°C in buffer III (buffer II supplemented with 0.1% NaDOC and 0.25% NLS) and resuspended in 1ml buffer III before being transferred to a TC12X12 AFA fiber tube (Covaris). Sonication was performed with an automated Covaris Focus ultra-sonicator with the following parameters: 20min, 5% duty cycle, 140W, 200 cycles and extracts were cleared by centrifugation 10min at 4°C. Chromatin sonication was evaluated on a Bioanalyzer DNA chip (Agilent) with one aliquot of material treated with RNAse A O/N at 65°C and purified with Qiagen minElute columns Total inputs and ChIPed samples were prepared for libraries using 10ng of material. Aliquots of the samples were tested in qPCR using the KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems, KK4604) to determine the optimal number of PCR cycles needed to amplify each library without reaching saturation. Libraries were amplified with KAPA HiFi Hotstart Readymix polymerase (KAPA Biosystems, KK2602), size-selected using Ampure XP beads (Beckman Coulter) and always quality checked on Bioanalyzer DNA high sensitivity chip (Agilent) and quantified by picogreen (Qubit® 2.0 Fluorometer, Invitrogen).