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| Status |
Public on Jul 27, 2017 |
| Title |
S9.6 DRIP Seq_2 |
| Sample type |
SRA |
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| Source name |
HeLa
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HeLa cells
sample type: DRIP
exogenous treatment: Control siRNA for luciferase
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| 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
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| 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.
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| 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).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| 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
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| Submission date |
Jun 14, 2017 |
| Last update date |
Sep 02, 2021 |
| Contact name |
Karlene Anne Cimprich |
| E-mail(s) |
cimprich@stanford.edu
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| Organization name |
Stanford University
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| Street address |
318 Campus Drive
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94025 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE93368 |
Genome wide DRIP-seq in control knockdown HeLa cells |
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| Relations |
| BioSample |
SAMN07237212 |
| SRA |
SRX2918366 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2668157_ip_gl3_rep2.bw |
195.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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