GSM3290784: LNCaP_N-Myc_DHT_H3K27me3_IP; Homo sapiens; ChIP-Seq
Cells were collected and crosslinked with 1% methanol-free formaldehyde for 10 minutes and fixation was quenched using 125 mM glycine for 8 minutes. The cell pellets were centrifuged and washed twice in cold PBS. Each pellet was resuspended in 1 ml of lysis buffer (50 mM Tris HCl pH 8, 0.5% SDS, 10 mM EDTA with protease and phosphatase inhibitors (Thermo Scientific)) and lysed for 20 minutes at 4°C. The nuclei were collected by centrifugation and resuspended in a second lysis buffer (10 mM Tris HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, with protease and phosphatase inhibitors (Thermo Scientific)). The protein-bound chromatin was sheared by sonication (Diagenode, Bioruptor Pico). Input sheared chromatin was reserved as a control for downstream ChIP-seq analysis. Equal volumes of sheared chromatin were immunoprecipitated with specified antibodies. Libraries were generated using the Hyper Prep Kit (Kapa Biosystems) following the manufacturer's recommended protocol.
GEO
GSM3290784