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| Status |
Public on Nov 05, 2015 |
| Title |
Input, treated, replicate 1 |
| Sample type |
SRA |
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| Source name |
MDA-MB-231
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231
cell type: triple negative breast adenocarcinoma
chip antibody: none (input)
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| Treatment protocol |
For conducting experiments, cells were grown to 80% confluency and serum starved overnight prior to treatment with 500uM DETA/NO for 24 hours.
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| Growth protocol |
MDA-MB-231 human triple negative breast cancer cells were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a controlled environment (37°C and 5% CO2).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
MDA-MB-231 cells in culture were crosslinked using 1% formaldehyde for 10 minutes at room temperature, following which excess reagent was quenched by the addition of 125 mM glycine for 5 minutes. Cells were washed with ice cold PBS and the pellet was resuspended in ChIP lysis buffer (1% SDS, 10 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.1, protease inhibitor cocktail and PMSF). DNA was sheared to 200-500bp using a Branson probe sonicator while keeping the sample on ice. A small aliquot of sheared chromatin was saved as “input”. Chromatin from 2 million cells was used for each ChIP reaction and diluted 10-fold using ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, protease inhibitor cocktail and PMSF). Immunoprecipitation was performed by incubating diluted chromatin with Dynabeads G (Invitrogen, pre-washed with BSA/PBS) and the following antibodies overnight at 4°C: H3K9me2 (Abcam, ab1220; 2 mg per ChIP) or H3K9ac (Millipore #07-352; 3.5 ml per ChIP). Beads were subsequently washed using the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiCl buffer (0.25M LiCl, 1% IGEPAL-CA630, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and twice with TE (1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Dissociation of chromatin-antibody complexes from the beads was facilitated by eluting twice using an appropriate buffer (1% SDS, 0.1M NaHCO3) for 15 minutes each at RT. Crosslinks in all ChIP and input samples were reversed by incubating with NaCl overnight at 65°C. Samples were treated with RNAse A for one hour at 37°C followed by addition of Proteinase K for one hour at 45°C for cleanup. In the final step, DNA was purified using the MinElute PCR Purification Kit (Qiagen).
Libraries were made using the Diagenode MicroPlex Library PreparationTM kit as per manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Sample name: Input_D1
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| Data processing |
[raw data QC and genome alignment] All raw sequencing data was quality trimmed to a minimum phred score of 20 using trimmomatic 0.32. Alignment to reference genome hg19 was done with BWA 0.7.5. PCR duplicates were removed using Picard MarkDuplicates and alignments with an edit distance greater than 2 to the reference, or that were mapped multiple times to the reference, were removed.
[ChIP-seq peak calling (H3K9ac)] Peak calls for H3K9ac data were created with Macs2 against paired input samples at an FDR threshold of 0.5 to allow for sufficient false peaks for IDR analysis; high-confidence peaks from individual replicates were selected using an FDR threshold of 1e-5 from the called peaks. Highly reproducible peaks were identified using Irreproducible Discovery Rate (IDR) using the IDR R package.
[ChIP-seq peak calling (H3K9me2)] Peak calls for H3K9me2 data were created with SICER against paired input samples using redundancy threshold = 10, window size = 500, fragment size = 400, effective genome fraction = 0.8, gap size = 1500, and an FDR threshold of 1e-5
Genome_build: hg19
Supplementary_files_format_and_content: bed file fields are: chromosome, start, end, peak ID, -log10 p-value or IDR value
Supplementary_files_format_and_content: bigWig files for H3K9ac (from Macs2) are raw read counts, bigWig files for H3K9me2 are normalized by library depth and background-subtracted
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| Submission date |
Feb 26, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Douglas David Thomas |
| E-mail(s) |
ddthomas@uic.edu
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| Phone |
312-996-6156
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| Organization name |
University of Illinois at Chicago
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| Department |
Medicinal Chemistry & Pharmacognosy
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| Street address |
900 S Ashland Ave Room 3306
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60607 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE66318 |
Nitric oxide regulates gene expression in cancers by controlling histone posttranslational modifications [HiSeq 2000] |
| GSE66324 |
Nitric oxide regulates gene expression in cancers by controlling histone posttranslational modifications |
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| Relations |
| BioSample |
SAMN03377059 |
| SRA |
SRX891836 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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