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Sample GSM4171652 Query DataSets for GSM4171652
Status Public on Nov 16, 2019
Title PP2_H3K27ac_rep1
Sample type SRA
Source name HUES 8 hESC line
Organism Homo sapiens
Characteristics cell line: HUES 8 hESC line
cell type: hPSC-derived pancreatic progenitor 2
Stage: Stage 4 day 5
chip antibody: H3K27ac (Active Motif: 39133)
Growth protocol Undifferentiated stem cells were maintained as aggregates in supplemented mTeSR1 medium (StemCell Technologies) using spinner flasks (Corning) set at a 70rpm rotation rate in a 37ºC 5% CO2 incubator. Directed hPSC differentiation into islet cells was conducted as described in the methods of (Alvarez-Dominguez et al. 2019).
Extracted molecule genomic DNA
Extraction protocol FACS-purified fixed cells (typically ~1million) were pelleted and flash-frozen. ChIP-seq was conducted as described in (Gifford et al., 2013) with minor modifications. Briefly, cell pellets were thawed on ice for 30min, incubated in lysis buffer (0.5% NP-40 +85mM KCl +20mM Tris-HCl pH8.0 +protease inhibitor) for 10min on ice, and nuclei were pelleted and incubated in lysis buffer (1% NP-40 +0.5% sodium deoxycholate +0.1% SDS +10mM Tris-HCl pH7.5 +protease inhibitor) for 10min on ice. Chromatin was then sheared with a Branson Sonifier (model S-450D) at 4ºC and incubated with 1µg/million cells H3K27Ac (Active Motif; 39133) or H3K4me1 (Millipore, 17-614) antibody overnight at 4ºC. Next, antibody-protein complexes were isolated by incubation with Protein A/G beads (Life Technologies; 100-02D/100-07D) for 2h at 4ºC. Samples were then sequentially washed twice with low-salt buffer (0.1% SDS +1% Triton X-100 +2mM EDTA +20mM Tris-HCl pH8.1 +150mM NaCl), twice with high-salt buffer (0.1% SDS +1% Triton X-100 +2mM EDTA +20mM Tris), twice with LiCl buffer (0.25M LiCl +1% NP-40 +1% deoxycholate +1mM EDTA +10mM Tris-HCl pH8.1), twice with TE (10mM Tris-HCl pH8.0 +1mM EDTA), and finally eluted in freshly-prepared elution buffer (1% SDS +0.1M NaHCO3) at 65ºC for 30min. Eluates were then treated with reverse crosslinking salt mixture (250mM Tris-HCl pH6.5 +62.5mM EDTA pH8.0 +1.25M NaCl +5mg/ml Proteinase K + 62.5ng/µl RNase A) overnight at 65ºC
DNA was purified using AMPure XP magnetic beads (Beckman Coulter), and sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs; E7103), pooled, and sequenced on a HiSeq 2500 instrument (Illumina).
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
Description PP2_SE.byH3K27ac.bed
Data processing Sequencing reads were mapped to the human hg19 reference genome assembly using bowtie2 (Langmead and Salzberg, 2012) with default parameters. Peak calling was performed using MACS2 (Zhang et al., 2008) with default parameters and “--broad --qvalue 0.01” to composite broad regions of read enrichment over background with a minimum FDR q-value cutoff of 0.01, using whole-cell extract from each stage as the background control. H3K4me1 and H3K27ac peaks across all cell types were concatenated and merged whenever overlapping by BEDTools (Quinlan and Hall, 2010) to obtain a unified catalog of H3K27ac/H3K4me1 regions (Figure S4) .
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited bed file containing enhancer domain coordinates
Submission date Nov 15, 2019
Last update date Nov 20, 2019
Contact name Juan R Alvarez-Dominguez
Organization name Harvard University
Department Stem Cell and Regenerative Biology
Street address 7 Divinity Ave
City Cambridge
State/province MA
ZIP/Postal code 02138
Country USA
Platform ID GPL16791
Series (2)
GSE139817 Chromatin dynamics reveal circadian control of human in vitro islet maturation
GSE140500 Chromatin dynamics reveal circadian control of human in vitro islet maturation [ChIP-seq]
BioSample SAMN13293885
SRA SRX7157634

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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