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| Status |
Public on Jan 25, 2019 |
| Title |
ChIP FOXA1_RR-HA |
| Sample type |
SRA |
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| Source name |
NIH-3T3
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| Organism |
Mus musculus |
| Characteristics |
cell line: NIH-3T3
cell type: mouse fibroblasts
chip antibody: Anti-HA.11 IgG (901501)
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| Treatment protocol |
NIH-3T3 cells were treated overnight with 500 ng/ml of Doxycycline one day after seeding. ChIP-seq was performed on at least 10 million cells.
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| Growth protocol |
NIH-3T3 cells were routinely cultured at 37°C and 5% CO2 in DMEM, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were grown in 100 mm cell culture dishes up to a confluence of 90% and split 1:6 every 3-4 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 10 minutes at room temperature and quenched with 200 mM Tris-HCl pH 8.0, washed with PBS, spun down, and stored at -80°C. The cell pellet was resuspended and in 1.5 ml LB1 (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X-100), incubated 10 min at 4°C, spun down, and resuspended in 1.5 ml LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) incubated 10 min at 4°C. The pellet was spun down, rinsed twice with SDS shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.15% SDS), and finally resuspended in 0.9 ml SDS shearing buffer. All buffers contain contain 1:100 diluted Protease Inhibitor Cocktail in DMSO (Sigma). The suspension was transferred to a milliTUBE 1 ml AFA fiber and sonicated on a E220 focused ultrasonicator (Covaris) using the following settings: 20 min, 200 cycles, 5% duty, 140W, and input sample aliquots were taken. To pull down HA-bound DNA, sonicated chromatin was incubated with anti-HA.11 antibody 16B12 (BioLegend) overnight at 4°C. 2.5 ml Protein G Dynabeads (Thermo Fischer) per one million cells were added to the chromatin and incubated 3 hours at 4°C. The chromatin was washed several times at 4°C with 5 min incubation between each wash and 2 min magnetization to collect beads; twice with Low Salt Wash Buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.15% SDS, 1 mM PMSF), once with High Salt Wash Buffer (10 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.15% SDS, 1 mM PMSF), once with LiCl Wash Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 250 mM LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM PMSF), and finally with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM PMSF). Beads were finally resuspended in Elution buffer (TE buffer with 1% SDS and 150 mM NaCl), treated with 400 ng/ml Proteinase K and reverse crosslinked at 65°C 1100 rpm overnight. Input samples were treated with 100 mg/ml RNase A and 400 ng/ml Proteinase K and reverse crosslinked at 65°C 1100 rpm overnight. Sonicated chromatin samples were purified using Qiagen MinElute PCR purification kit.
Libraries were prepared with NEBNext ChIP-seq Library Prep Master Mix kit with insert size selection of 250 bp.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Overexpression of FOXA1_RR-HA
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| Data processing |
Reads were aligned to the mouse reference genome mm10 using STAR 2.5.3a with settings ‘-- alignMatesGapMax 2000 --alignIntronMax 1 --alignEndsType EndtoEnd -- outFilterMultimapNmax 1’
Duplicate reads were removed with Picard and reads not mapping to chromosomes 1-19, X, or Y were removed
Peaks were called with MACS 2.1.1.20160309 with settings ‘-f BAMPE -g mm’. Peaks overlapping peaks called for input (non-immunoprecipitated chromatin) from NIH- 3T3 cells and ENCODE blacklisted peaks were discarded.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files were generated with deepTools 2.4.2 with settings '--normalizeUsingRPKM'
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| Submission date |
Sep 11, 2018 |
| Last update date |
Jan 27, 2019 |
| Contact name |
David Michael Suter |
| Organization name |
École polytechnique fédérale de Lausanne
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| Department |
Institute of Bioengineering
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| Lab |
UPSUTER
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| Street address |
EPFL SV-IN Station 19
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| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE119782 |
Mitotic chromosome binding predicts transcription factor properties in interphase [ChIP-Seq] |
| GSE119784 |
Mitotic chromosome binding predicts transcription factor properties in interphase |
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| Relations |
| BioSample |
SAMN10032334 |
| SRA |
SRX4669316 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3383716_FOXA1_RR.bw |
154.5 Mb |
(ftp)(http) |
BW |
| GSM3383716_FOXA1_RR_filtered.bed.gz |
1.3 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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