GSM7696003: hiPSC H3K9me3 ChIP-Seq rep1; Homo sapiens; ChIP-Seq
Cells were fixed with 0.8% methanol-free formaldehyde in DMEM for 10 min, followed by quenching with 0.125 M glycine for 5 min. Cells were resuspended in Farnham buffer (5 mM PIPES, pH 8; 85 mM KCl; 0.5% IGEPAL-CA 630), followed by snap freezing in liquid nitrogen. All buffers were supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail before use. Chromatin was isolated and sheared following the NEXSON protocol. Frozen cell pellets were thawed on ice and sonicated for 2 min in 1- ml tubes on a Covaris S220 with the following settings: peak power = 75W; duty factor = 2%; cycles/burst = 200. Isolated nuclei were washed with Farnham buffer once and resuspended in shearing buffer (10 mM Tris-HCl, pH 8; 0.1% SDS; 1 mM EDTA). Chromatin was sheared on a Covaris S220 by sonication for 18 min in 1-ml tubes with the following settings: peak power = 140W; duty factor = 5%; cycles/burst = 200. Sheared chromatin was snap frozen in liquid nitrogen, aliquoted and stored at -80°C. Sequencing libraries from ChIP eluted DNA samples were prepared with the Ovation® Ultralow V2 DNA-Seq Library Preparation Kit (TECAN, #0344NB) and SPRIselect beads (Beckman Coulter, #B23318) according to the manufacturer's protocol. 110-bp paired-end sequencing was performed on a NovaSeq platform (Illumina).