title: GSM1940201: K562 NaBut PU.1 ChIP-seq Rep 1; Homo sapiens; ChIP-Seq
library_construction_protocol: Perfomed as described in PMID:17540862. Chromatin sonicated with Bioruptor (Diagenode) for 45 min.( 30 sec. on/off, High) in 1x RIPA buffer. DNA fragments resuspended in 1% Triton X-100, 2mM EDTA, 20mM TrisCl, 150 mM NaCl buffer, pre-cleared with Protein A Dynabeads for 2 hr. at 4°C, and 6 μg of rabbit anti-PU.1 Santa Cruz Biotech sc-22805 (Life Tech.) incubated with the sonicated chromatin overnight at 4°C. Protein A beads were bound to antibody-chromatin mixture for 3 hours at 4°C and then washed 5 times with LiCl wash buffer (500 mM LiCl, 100 mM TrisCl, 1% NP40, 1% sodium deoxycholate) and once with 1x TE . Crosslinking was reversed with overnight 65°C incubation in 1% SDS, 0.1M sodium bicarbonate buffer and DNA was eluted with a PCR cleanup kit (Qiagen). Following TruSeq adapter ligations, DNA fragments were separated from free adapters by AmpureXL bead size selection steps and PCR amplification was performed for 18 cycles. ~20 million cells crosslinked with 1% formaldehyde for 10 min. at RT, quenched with glycine, and lysed with 1x RIPA buffer.
identifiers.namespace: GEO
identifiers.id: GSM1940201