title: GSM4774318: s167FLAGrep1 ChIP-seq; Homo sapiens; ChIP-Seq
library_construction_protocol: Cultured CTCs were washed with ice-cold phosphate buffered saline (PBS) and homogenized while on ice using lysis buffer (0.32M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM EDTA, 10 mM Tris-HCL (pH 8.0), 1 mM DTT and 0.1% Triton X-100). The lysate was crosslinked using 1% formaldehyde for 10 min at room temperature and then quenched with glycine. ChIP was performed using Magna ChIP(TM) Hisens Chromatin Immunoprecipitation Kit (Millipore Cat#17-104-60) following the manufacturer's instructions. Briefly, the precipitated nuclei were resuspended in SCW buffer containing protease inhibitor cocktail, followed by sonication using a Covaris S2, yielding an average fragment size of 250bp. The chromatin was then immunoprecipitated using Protein A Dynabeads (In vitrogen) with 5mg of ChIP-grade antibody overnight at 4oC. The beads were repeatedly washed with high- and low-salt buffers, and the DNA fragments were then de-crosslinked using proteinase K at 65oC for 4 hrs, followed by ethanol precipitation. ChIP-seq library was constructed using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (Catlog# E7645L).