title: GSM2551205: CPC H3K4me3 rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: Day 5 cardiac progenitor cells, C-ECs, and H-ECs were fixed in 1% formaldehyde for 10 minutes at RT to crosslink the chromatin.  Following that glycine was added to a final concentration of 0.125 M. Cells were washed twice with cold PBS and frozen at -80ºC. Crosslinked samples were submitted to the University of Washington High Throughput Genomic Sequencing Center for ChIP-seq analysis for H3K4me3 and H3K27me3 histone modifications. Total RNA from bulk cultures of cardiac progenitor cells, C-ECs, and H-ECs were isolated with RNALater (Qiagen, 76104). For each group, 2 biological replicates were submitted for analysis. The chromatin was sonicated to small fragments (<500 bp) and histone bound chromatin was immunoprecipitated using antibiodies specific to H3K4me3 (Cat # 9751, Cell Signaling) or H3K27me3 (Cat # 07-449; Millipore). For each IP, Dynabeads (M-280, sheep anti-rabbit IgG, Invitrogen) were incubated with antibodies for 6 hr at 4°C and then incubated overnight with ∼100 μg sheared chromatin. The complexes were rinsed sequentially with IP wash buffer I (50 mMTris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA [pH8.0], 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mMTris-HCl pH8.0, 0.5 M NaCl, 1 mM EDTA [pH8.0], 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), IP wash buffer II (50 mMTris-HCl [pH8.0], 1 mM EDTA pH8.0, 1% NP-40, 0.7% sodium deoxycholate, 0.5 M LiCl), and TE buffer (10 mMTris-HCl [pH8.0], 1 mM EDTA pH8.0). The complexes were incubated with elution buffer (10 mMTris-HCl [pH 8.0], 0.3 M NaCl, 5 mM EDTA [pH8.0], 0.5% SDS) supplemented with RNase A (Ambion) at 65°C overnight. After separation, the DNA was treated with Proteinase K and purified by using PCR purification column (QIAGEN). The Illumina indexed sequencing libraries were prepared following a standard protocol using PE adapters (Illumina). The libraries were used to generate sequencing clusters on flowcells using cBot, and sequenced on Illumina HiSeq 2000 to generate PE 2X36 base pair reads.  Each library was on average sequenced to a depth of ∼18 million tags that mapped uniquely to the human genome (hg19). Samples were submitted to University of Washington High Throughput Genomic Sequencing Center for isolation and analysis. RNA-seq was performed on poly-A enriched samples using Illumina TruSeq.
attributes.tag: GEO Accession
attributes.value: GSM2551205