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Sample GSM2897180 Query DataSets for GSM2897180
Status Public on Apr 30, 2019
Title ChIP-seq_HAP1_WT_BRD4_r1
Sample type SRA
Source name HAP1 cell line
Organism Homo sapiens
Characteristics library: ChIP-seq
cell line: HAP1
replicate: 1
ip: BRD4
clone: C631
Growth protocol HAP1 WT and knock-out cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Thermo Fisher Scientific, 21980-032) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS, Thermo Fisher Scientific, 10500) and 1% Pen Strep (100 units/ml Penicillin, 100 µg/ml Streptomycin, Thermo Fisher Scientific, 15140-122). A549 cells were cultured in F-12K Nut Mix (Thermo Fisher Scientific, 21127-022) supplemented with 10% FBS and 1% Pen Strep.
Extracted molecule genomic DNA
Extraction protocol ChIPmentation was carried out as described (Schmidl et al, Nature Methods 2015). Cells were washed once with PBS and fixed with 1% paraformaldehyde in up to 1ml PBS for 10 min at room temperature. Glycine was added to stop the reaction. Cells were collected at 500 g for 10min at 4 C, washed twice with cold PBS supplemented with 1 mM phenylmethyl sulfonyl fluoride (PMSF). The pellet was lysed in sonication buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 0.25% SDS, 1uL protease inhibitors (Sigma) and 1 mM PMSF) and sonicated with a Covaris S220 sonicator to a range of 200-700 bp. Lysates were centrifuged at full speed for 5min at 4 C, and the supernatant transferred to a new tube. The lysate was then brought to RIPA buffer conditions (final concentration: 10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 140mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1uL protease inhibitors (Sigma) and 1 mMPMSF) to a volume of 200 ml per IP. For each immunoprecipitation, 10 ml magnetic Protein A (Life Technologies) were washed twice and resuspended in PBS supplemented with 0.1% BSA. The antibody was added and bound to the beads by rotating 2 h at 4 C. Used antibodies were H3K4me1 (0.5 mg per IP, Diagenode pAb-194-050), H3K27ac (1 mg per IP, Diagenode pAB-196-050) and H3K27me3 (1 mg per IP, Millipore 07-499). For control libraries, an IP with 2.5 mg of a nonspecific IgG rabbit antibody was used. Blocked antibody-conjugated beads were then placed on a magnet, supernatant was removed and the sonicated lysate was added to the beads followed by incubation for 3-4 h at 4 C on a rotator. Beads were washed subsequently with RIPA (twice), RIPA-500 (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 500mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% DOC) (twice) and RIPA-LiCl (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 250mM LiCl, 1% Triton X-100, 0.5% DOC and 0.5% NP40) (twice). Beads were washed once with cold Tris-Cl pH 8.0, to remove detergent, salts and EDTA. Beads were washed once more with cold Tris-Cl pH 8.0.
DNA was purified with SPRI AMPure XP beads (sample-to-beads ratio 1:2) or Qiagen MinElute columns. One microlitre of each library was amplified in a 10-ml qPCR reaction containing 0.15 mM primers, 1uL SYBR Green and 5 ml Kapa HiFi HotStart ReadyMix (Kapa Biosystems), to estimate the optimum number of enrichment cycles with the following programme: 72 oC for 5min, 98 oC for 30 s, 24 cycles of 98 oC for 10 s, 63 oC for 30 s and 72 oC for 30 s, and a final elongation at 72 oC for 1min. Kapa HiFi HotStart ReadyMix was incubated at 98 oC for 45 s before preparation of all PCR reactions (qPCR and final enrichment PCR). Final enrichment of the libraries was performed in a 50-ml reaction using 0.75 mM primers and 25 ml Kapa HiFi HotStart ReadyMix. Libraries were amplified for N+1 cycles, where N is equal to the rounded-up Cq value determined in the qPCR reaction. Enriched libraries were purified using SPRI AMPure XP beads at a beads-to-sample ratio of 1:1, followed by a size selection using AMPure XP beads to recover libraries with a fragment length of 200-400 bp. Library preparation was performed using custom Nextera primers as described for ATAC-seq. The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 4000 platform and the 50-bp single-end configuration.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 4000
Description ChIP-seq for BRD4 on HAP1 cell line, replicate 1
Data processing Base calls provided by the Illumina Realtime Analysis software were converted into BAM format using Illumina2bam and demultiplexed using BamIndexDecoder (
Sequenced reads were trimmed for adaptor sequences
Reads were mapped to hg19 whole genome using bowtie2 v2.2.4 with the –very-sensitive parameter
Duplicate reads were marked and removed with picard tools version 1.118
Library quality was assessed with the phantomPeakQualtools scripts (Landt SG, Genome Research 2012).
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files contain counts of reads per basepair
Supplementary_files_format_and_content: csv file contains read counts for each sample in each regulatory element
Submission date Dec 21, 2017
Last update date May 07, 2019
Contact name Christoph Bock
Organization name CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
Street address Lazarettgasse 14
City Vienna
ZIP/Postal code 1090
Country Austria
Platform ID GPL20301
Series (2)
GSE108387 Systematic functional characterization of BAF mutations yields novel intra-complex synthetic lethalities [ChIP-Seq]
GSE108390 Systematic functional characterization of BAF mutations yields novel intra-complex synthetic lethalities
BioSample SAMN08224243
SRA SRX3503209

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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