 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 24, 2021 |
| Title |
ChIP on NELFA and with no estradiol treatment of MCF-7 cells [NELFA_minusE2_Fan150] |
| Sample type |
SRA |
| |
|
| Source name |
ChIP on NELFA and with no estradiol treatment of MCF-7 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
chip antibody: NELFA
|
| 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.
|
| Growth protocol |
MCF-7 cells were cultured in DMEM, 10% FBS and 1% penicillin-streptomycin in a humidified incubator with 5% CO2 at 37C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as previously described, with minor modifications. Briefly, approximately 5 x 10^7 cells were crosslinked with 1% formaldehyde at room temperature for 15 min and quenched with 0.125M glycine for 5 min. Cells were then harvested and incubated with nuclei preparation buffer (50 mM Tris pH 7.5, 150 mM NaCl , 5 mM EDTA, 0.5% NP-40, 1% TX-100), with protease inhibitor (Sigma, Cat#P2714-1BTL) included, for 10 min. After centrifugation, the pellet was suspended in sonication buffer (10 mM Tris, pH8.0, 100 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate and 0.5% N-lauroylsarcosine, with protease inhibitor included) for sonication. After sonication, 1/10 volume of 10% Triton X-100 was added to sonicated lysate. The supernatant, after centrifugation, was then incubated with 5μg of antibody at 4°C overnight. Immunoprecipitated complexes were collected using Protein G magnetic beads (Invitrogen, Cat#10004D). Immuno-complexes were washed with RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0.7% Na-Deoxycholate, with protease inhibitor included) for at least 5 times and TE buffer containing 50 mM Nacl once. After de-crosslinking, DNA was then purified by QIAquick Spin columns (Qiagen).
For Bio-ChIP, 5x10^7 MCF-7 cells stably expressing BirA and biotin acceptor peptide (BAP)-HP1α (4A) or BAP- HP1α (4E) were crosslinked with 1% formaldehyde at room temperature for 15 min, and then incubated with 0.125M glycine for 5 min. To isolate nuclei, cells were incubated with nuclei preparation buffer (50 mM Tris pH 7.5, 150 mM NaCl , 5 mM EDTA, 0.5% NP-40, 1% TX-100), with protease inhibitor (Sigma, Cat#P2714-1BTL) included, for 10 min. The obtained nuclei were 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 sonicated chromatin in immunoprecipitation buffer was then incubated with 50 ul of pre-blocked Pierce™ Streptavidin Magnetic Beads (Thermo Fisher, Cat#88816) overnight at 4°C on a rotating wheel. Beads were extensively 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. Beads were then treated with RNase A (Thermo Fisher Scientific, Cat# AM2270) and Proteinase K (Thermo Fisher Scientific, Cat# AM2548) before de-crosslinking overnight at 65°C. DNA was finally purified by QIAquick Spin columns (Qiagen, Cat#28106).
The library construction protocol followed the manufacturer’s instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
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 CHIP-seq analysis, we have used the default settings in HOMER.
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
|
| |
|
| Submission date |
Oct 21, 2019 |
| Last update date |
Mar 24, 2021 |
| Contact name |
MICHAEL G ROSENFELD |
| E-mail(s) |
[email protected]
|
| Organization name |
HHMI/UCSD
|
| Street address |
9500 Gilman Drive
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE139196 |
ChIRC-seq Reveals Promoter Antisense RNAs and HP1α as Key Regulators of Promoter Pause Release and of Enhancer : Architectural RNA Interactions [ChIP-Seq] |
| GSE139199 |
Shape of promoter antisense RNAs regulates ligand-induced transcription activation |
|
| Relations |
| BioSample |
SAMN13071138 |
| SRA |
SRX7030928 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4133840_NELFA_minusE2.Fan150_S17.Fan_150_S17_L005_R1_001.fastq.gz.after.trimmomatic.fastq.gz.bed.srm.dir.ucsc.bedGraph.gz |
134.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
