GSM1327871: AR ChIP-seq shCtrl R1881, biological replicate 2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
chip antibody vendor
Sequenced DNA Library
Antibodies were from abcam (cat#ab23738,GR77830-1) and Millipore (cat#06-680, lot#JBC1939961) and ChIP was performed using 5e6-10e6 cells per reaction. Chromatin was fixed in room temperature for 10 minutes with serum free media containing 0,37 % formaldehyde. Cells were washed twice in PBS and scraped before treatment of cell lysis buffer with protease inhibitors on ice. Pelleted nuclei were sheared to 200-700bp using an ultrasonic sonicator (Misonix). ChIP was done with 5 micrograms of antibody. FAIRE analysis was performed according to the protocol published by Giresi et al . Libraries were prepared according to Bioo Scientific's instructions accompanying the DNA Sample Kit (Part# 514101). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiScan following the manufacturer's protocols.