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| Status |
Public on Mar 24, 2021 |
| Title |
ChIRC on 7SK and with no estradiol treatment of MCF-7 cells FanC213 |
| Sample type |
SRA |
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| Source name |
ChIRC on 7SK and with no estradiol treatment of MCF-7 cells FanC213
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
complex: 7SK
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| Treatment protocol |
For estrogen stimulation, these cells were cultured in phenol-red free DMEM with 5% charcoal stripped FBS for at least 3 days and then treated with 100nM 17β-estradiol (E2) for 40 minutes. Control samples were treated with ethanol.
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| Growth protocol |
MCF-7 cells were cultured in DMEM, 10% FBS and 1% penicillin-streptomycin in a humidified incubator with 5% CO2 at 37C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
All the experiments were performed in RNase-free environment and every buffer used in the experiment was supplemented with SUPERase• In™ RNase Inhibitor (Thermo Fisher Scientific, Cat#AM2696). Briefly, 5x10^7 cells stably expressing biotin acceptor peptide (BAP)-dCas13a, BirA and corresponding gRNA were crosslinked with 1% formaldehyde at room temperature for 15 min before being quenched with 0.125M glycine for 5 min. Cells were first incubated in Lysis buffer 1 (10 mM Tris pH 8, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, protease inhibitor included) for 10 min on ice and then were incubated in Lysis buffer 2 (10 mM Tris pH 8, 1 mM EDTA, 0.5 mM EGTA and 200 mM NaCl, protease inhibitor included) for 5 min on ice for nuclei isolation. After centrifugation, the pellet was suspended in immunoprecipitation buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate and 1% Triton X-100, with protease inhibitor included) for sonication. The supernatant, after centrifugation, was then incubated with 50 ul of pre-blocked Pierce™ Streptavidin Magnetic Beads (Thermo Fisher, Cat#88816) at 4°C overnight. Beads were then washed with SDS buffer (10 mM Tris, pH 8, 1 mM EDTA, 2% SDS, protease inhibitor included) twice, high salt buffer (50 mM HEPES pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate and 500 mM NaCl) twice, DOC buffer (250 mM LiCl, 0.5%, NP-40, 0.5% deoxycholate, 1 mM EDTA and 10 mM Tris pH 8) once and TE buffer containing 50 mM Nacl once. After RNase A, Proteinase K treatment and de-crosslinking, DNA was then purified by QIAquick Spin columns (Qiagen)
For ChIRC-seq, the extracted DNA was ligated to specific adaptors followed by deep sequencing with the Illumina's HiSeq 2500 system according to the manufacturer's instructions.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Library strategy: ChIRC-seq
The image analysis and base calling were performed by using Illumina's Genome Analysis pipeline.
Quality control: FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and TRIMMOMATIC (http://www.usadellab.org/cms/?page=trimmomatic) was used to inspect the quality of the sequencing reads, and to trim the sequencing adaptors, if necessary.
Read alignment: Bowtie2 (Langmead et al., Nature Methods 2012) was employed to align ChIP-seq and ChIRC-seq samples to hg38 genome and we have kept 1 copy/read per genome position.
Peak finding: we have called the ChIP-seq and ChIRC-seq peaks by using HOMER findPeaks subroutine (Heinz et al., 2010, http://homer.ucsd.edu/homer/). For the ChIRC-seq analysis, we have used the input samples in order to estimate the background, and the default settings were adjusted to order to capture the biology of ncRNA binding to the genome.
Generation of bedgraph files: we have used the HOMER scripts makeUCSCfile and makeMultiWigHub.pl in order to generate bedgraph files for visualization in the UCSC genome browser.
Genome_build: hg38
Supplementary_files_format_and_content: bedgraph
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| Submission date |
Oct 21, 2019 |
| Last update date |
Mar 24, 2021 |
| Contact name |
MICHAEL G ROSENFELD |
| E-mail(s) |
[email protected]
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| Organization name |
HHMI/UCSD
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| Street address |
9500 Gilman Drive
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE139137 |
ChIRC-seq Reveals Promoter Antisense RNAs and HP1α as Key Regulators of Promoter Pause Release and of Enhancer : Architectural RNA Interactions [ChIRC-seq] |
| GSE139199 |
Shape of promoter antisense RNAs regulates ligand-induced transcription activation |
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| Relations |
| BioSample |
SAMN13067056 |
| SRA |
SRX7026895 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4131782_ChIRC_7SK_gRNA3_minusE2_FanC213.Fan_C213_S10_L005_R1_001.fastq.gz.after.trimmomatic.fastq.gz.bed.srm.dir.ucsc.bedGraph.gz |
154.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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