title: GSM1483892: input; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were fixed in 1% formaldehyde solution for 8 min followed by quenching of the reaction with glycine (125 mM) for 5 min. Cell were lysed in ChIP Lysis Buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1) and sonicated using Bioruptor Pico (Diagenode). Sonication was performed at 4°C using 5 sets of 10 cycles (each consisting of 30s sonication, 30s refraction). Protein A Sepharose beads (Sigma, P9424) were washed and pre-blocked in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167mM NaCl, 16.7 mM Tris-HCl pH 8.1) with 5mg/ml BSA and 0.2mg/ml yeast tRNA (Sigma, R5636) for 2 hours at 4°C.  Once sonication was complete, samples were diluted 10 times with ChIP dilution buffer and incubated with pre-blocked beads for 1 hour. Beads were then removed and the pre-cleared samples were incubated overnight with 2µg/sample of anti-H3K4me3 (Millipore, 07-473) at 4°C. Beads were washed twice with ChIP Low Salt Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl pH 8.1), once with ChIP High Salt Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, 20mM Tris-HCl pH 8.1), once with ChIP LiCl Buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1) and twice with TE buffer (1mM EDTA, 10mM Tris-HCl pH 8.0). Chromatin was eluted with ChIP Elution buffer (1% SDS, 100mM NaHCO3, 250mM NaCl) and de-cross-linked at 65°C for at least 4 hours. Protein was digested by adding 0.7mg/ml Proteinase K and heating at 55°C for at least 1 hour. DNA was isolated using 1.8 volumes of AMPureXP beads (Beckman Coulter, A63881). DNA libraries for Next-Generation Sequencing (NGS) were prepared using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB, E7370S) and NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) (NEB, E7335S). Samples were 4-plexed on each lane and 50 bp single-ended reads were obtained.
attributes.tag: GEO Accession
attributes.value: GSM1483892