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| Status |
Public on May 13, 2021 |
| Title |
Input on A673 STAG2 WT matched for H3K27Ac |
| Sample type |
SRA |
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| Source name |
A673 clones with STAG2 wild type
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| Organism |
Homo sapiens |
| Characteristics |
cell line: A673 Ewing sarcoma
genotype/variation: STAG2 wild-type
chip antibody: Input matched for H3K27Ac
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| Growth protocol |
Chromatin immunoprecipitation sequencing for EWS/FLI, H3K27Ac, and CTCF: Adherent A673 cells with and without STAG2 knockout (sgNT-1c4, sgNT-2c3, sgSTAG2-1c6, and sgSTAG2-4c5) were grown to 70-80% confluence, crosslinked with 1% formaldehyde for 5 min and then quenched with 0.125 M glycine. The fixed cells were harvested with trypsin and washed twice in ice cold PBS before resuspension in lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS) supplemented with protease inhibitors (Roche). Chromatin immunoprecipitation sequencing for SMC1a: SMC1a ChIP was performed as previously described with a few adaptations. Adherent A673 cells with and without STAG2 knockout (sgNT-1c4, sgNT-2c3, sgSTAG2-1c6, and sgSTAG2-4c5) were crosslinked for 15 minutes at room temperature by the addition of one-tenth volume of fresh 11% formaldehyde solution (11% formaldehyde, 50 mM HEPES pH 7.3, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) to the growth media followed by 5 min quenching with 125 mM glycine. Cells were rinsed twice with 1X PBS and harvested using a silicon scraper and flash frozen in liquid nitrogen. Frozen crosslinked cells were stored at -80°C. 100µl of Protein G Dynabeads (Life Technologies #10009D) were washed 3X for 5 minutes with 0.5% BSA (w/v) in PBS at 4oC. Magnetic beads were bound with 10 µg of anti-SMC1 antibody (Bethyl A300-055A) overnight at 4°C, and then washed 3X with 0.5% BSA (w/v) in PBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation sequencing for EWS/FLI, H3K27Ac, and CTCF: Chromatin was sheared to about 200 bp fragments using the Covaris ultra-sonication. The lysate was diluted with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.1, 16.7 mM NaCl, 1.2 mM EDTA, 1.1% Triton-X100, 0.01% SDS) and cleared by centrifugation. 5% of the supernatant volume set aside as an input control. The remaining lysate was incubated over night at 4°C with 5 ug of antibody (3 ug for histone antibodies). The following antibodies were used for ChIP: Fli (sc-356X, Santa Cruz Biotechnology), H3K27Ac (ab4729, Abcam), and CTCF (07-729, EMD Millipore). Chromatin immunoprecipitation sequencing for SMC1a: Cells were prepared for ChIP as follows. All buffers contained freshly prepared 1× cOmplete protease inhibitors (Roche, 11873580001). Frozen crosslinked cells were thawed on ice and then resuspended in lysis buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1× protease inhibitors) and rotated for 10 minutes at 4°C, then spun at 1350 rcf for 5 minutes at 4°C. The pellet was resuspended in lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease inhibitors) and rotated for 10 minutes at 4°C and spun at 1350 rcf for 5 minutes at 4°C. The pellet was resuspended in sonication buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, and 1% Triton X-100, 1× protease inhibitors) and then sonicated on a Misonix 3000 sonicator for 10 cycles at 30 seconds each on ice (18-21 W) with 60 seconds on ice between cycles. Sonicated lysates were cleared once by centrifugation at 16,000 rcf for 5 minutes at 4°C. 50 µL was reserved for input, and then the remainder was incubated overnight at 4°C with magnetic beads bound with antibody to enrich for DNA fragments bound by the indicated factor.
Chromatin immunoprecipitation sequencing for EWS/FLI, H3K27Ac, and CTCF: The precipitated complexes were bound to protein A/G high-capacity agarose beads (Pierce) for 2 h before washing sequentially with ice cold low salt wash (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100, 0.1% SDS), high salt wash (20 mM Tris-HCl pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton-X100, 0.1% SDS), LiCl wash (10 mM Tris-HCl pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% deoxycholic acid, 1% IGEPAL CA-630) and TE wash (10 mM Tris-HCl pH 8.1, 1 mM EDTA) and eluted in elution buffer (0.1 M NaHCO3, 1% SDS). Library preparation for eluted DNA fragments were performed using the NEBNext ChIP-Seq Library Prep for Illumina (New England Biolabs). The Pippin Prep (Sage Science) was used to size select the adaptor ligated DNA in the 175-450 bp range prior to PCR enrichment. Libraries were sequenced on the Illumina HiSeq 2000 instrument. Chromatin immunoprecipitation sequencing for SMC1a: For ChIP-seq experiments, purified ChIP DNA was used to prepare Illumina multiplexed sequencing libraries.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Quality control tests for unmapped sequences were performed based on the FastQC v.0.11.5 software (Babraham Bioinformatics, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
ChIP-seq reads were aligned to the hg19 genome assembly using bwa v0.7.17 with the mem -M configuration. Quality control tests for the mapped reads were performed by using the ChIPQC library available from Bioconductor v3.5.
Data were filtered for duplicate reads with Picard tools.
Peak calling was performed using the model-based MACS v1.4.3 software, with the cut-offs P ≤ 1e-09 for H3K27Ac and P ≤ 1e-05 for all other marks. The ENCODE black-listed regions for hg19 (available at https://www.encodeproject.org/annotations/ENCSR636HFF/) were removed from each set of peak regions.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Bigwig files for normalized coverage signal in units of reads per million mapped reads per bp (rpm/bp). Coverage was computed for bins of size 20 base pairs by using the bamCoverage module available in deepTools v2.5.3.
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| Submission date |
Jul 02, 2018 |
| Last update date |
May 15, 2021 |
| Contact name |
Gabriela Alexe |
| E-mail(s) |
[email protected]
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| Organization name |
Broad Institute
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| Department |
Computational Biology and Bioinformatics
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| Street address |
415 Main St.
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE116495 |
The effect of STAG2 loss in Ewing sarcoma |
| GSE165783 |
The effect of STAG2 loss in Ewing sarcoma [ChIP-seq] |
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| Relations |
| BioSample |
SAMN09532151 |
| SRA |
SRX10889821 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3240321_A673.STAG2WT.Input_H3K27Ac.bw |
426.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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