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Sample GSM2668157 Query DataSets for GSM2668157
Status Public on Jul 27, 2017
Title S9.6 DRIP Seq_2
Sample type SRA
Source name HeLa
Organism Homo sapiens
Characteristics cell type: HeLa cells
sample type: DRIP
exogenous treatment: Control siRNA for luciferase
Treatment protocol HeLa cells were transfected with siRNA against luciferase (Fisher scientific D-001400-01-20) at 20 nM for 72 hours using Dharmafect, according to manufacturer protocols
Growth protocol HeLa cells were cultured in DMEM (GIBCO) supplemented with 10% FBS, 2 mM L-glutamine and penicillin/streptomycin in 5% CO2 at 37°C.
Extracted molecule genomic DNA
Extraction protocol DRIP was performed as described in Ginno et al., 2012. Briefly, DNA was extracted with phenol/chloroform in phase lock tubes (5Prime), precipitated with EtOH/sodium acetate, washed with 70% EtOH, and resuspended in TE. DNA was digested with EcoRI, XbaI, SspI, BsrgI, and HindIII (NEB) restriction enzymes overnight at 37 °C. For RNase H-treated samples, 4 ug of DNA was treated with RNase H (NEB, M0297S) overnight at 37 °C. DNA was purified by phenol/chloroform, EtOH/sodium acetate precipitation as described above. 4μg of DNA was bound with 10 μg of S9.6 antibody in 1 X binding buffer (10 mM NaPO4 pH 7, 140 mM NaCl, 0.05% Triton X-100) overnight at 4°C. Protein A/G sepharose beads (Pierce) were added for 2 h. Bound beads were washed 3 times in binding buffer and elution was performed in elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, Proteinase K) for 45 min at 55°C. DNA was purified as described. In addition, sheared Drosophila melanogaster chromatin (Active Motif, 53083) and a Drosophila-specific H2Av antibody (Active Motif, 61686) was spiked-in as a minor fraction of the DRIP DNA to allow normalization of read counts according to the manufacturer’s protocol.
Using asynchronously growing HeLa cells, DRIP followed by library preparation, next generation sequencing, and peak calling were performed as described in Stork et al., 2016 with minor modifications. RNase A pre-treatment before DRIP was conducted with 6 μg/ml of enzyme for 45 min at 37ºC in 10 mM Tris-HCl pH 7.5 supplemented with 0.5M NaCl as described in Sanz et al., 2016 . RNase A pre-treatment before DRIP was conducted with 6 μg/ml of enzyme for 45 min at 37ºC in 10 mM Tris-HCl pH 7.5 supplemented with 0.5M NaCl as described in Sanz et al., 2016 . Libraries were pooled and sequenced on an Illumina HiSeq 4000 machine with paired-end 75bp reads at the Stanford Functional Genomics Facility (NIH grant S10OD018220).
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 4000
Data processing Base calling CASAVA v1.8.2
Reads were aligned to genome with bowtie2 -p 9 --qc-filter --no-unal --no-mixed --no-discordant . The used reference genome was a concatenation of hg19 to the drosophilla genome r5.37 from FlyBase.
Reads mapping exclusively to the drosophilla genome were filtered out - only reads mapping to hg19 were kept for further processing.
Reads processed into bedpe format using bedtools
Reads sorted and filtered to only include those with mappability score >10 using unix shell utilities
Generate bigwig files using bedtools coverageBed and bedGraphToBigWig from UCSC
Genome_build: hg19
Supplementary_files_format_and_content: bigwig
Submission date Jun 14, 2017
Last update date Sep 02, 2021
Contact name Karlene Anne Cimprich
Organization name Stanford University
Street address 318 Campus Drive
City Stanford
State/province CA
ZIP/Postal code 94025
Country USA
Platform ID GPL20301
Series (1)
GSE93368 Genome wide DRIP-seq in control knockdown HeLa cells
BioSample SAMN07237212
SRA SRX2918366

Supplementary file Size Download File type/resource 195.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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