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| Status |
Public on Mar 06, 2017 |
| Title |
ZNF540 |
| Sample type |
SRA |
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| Source name |
transduced 293T cell line
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| Organism |
Homo sapiens |
| Characteristics |
host cell line: HEK 293T
tagged protein: ZNF540
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| Treatment protocol |
Transduced cell lines were selected using puromycin (1 ug/mL) for 7 days and induced using doxycyclin (1 ug / mL) for a few days before fixation with formaldehyde (1% for 10 min).
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| Growth protocol |
Human embryonic kidney-derived 293T cells were maintained in Dulbecco's modified Eagle's complete medium supplemented with 10% fetal bovine serum, 0.29 mg/ml L-glutamine, and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO2. Human embryonic stem cells (Line H1) were cultured in mTeSR1 medium (Stem Cell Technologies) on Matrigel (BD Biosciences) and in the presence of ROCK inhibitor (Y-27632, Stem Cell Technologies).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared by resuspending 3x107 293T cells in 6 mL of PBS, adding 375 µL of methanol-free formaldehyde (16% - 1% final) and incubating for 10 min on a rotating wheel, quenching with Tris pH 8.0 (250 mM final) for 10 min on a rotating wheel, rinsing with PBS twice and freezing the pellet at -80°C. Nuclear extraction was performed as follow: fixed cell pellets were thawed on ice and resuspended in 1.5 mL of LB1 buffer (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP40, 0.25% Tx100) and incubated for 10 min at 4°C on a rotating wheel. The mixture was centrifuged at 1,700g at 4°C, the supernatant removed, the pellet resuspended in 1 mL LB2 buffer (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and incubated for 10 min at 4°C on a rotating wheel. The same procedure was repeeated 2 more times, replacing LB2 with SDS shearing buffer (10 mM Tris pH 8.0, EDTA 1 mM, SDS 0.15%) and the pellet was finally resuspended in 1 mL of this last buffer. Every buffer was prechilled and contained a protease inhibitor cocktail (cOmplete ULTRA Tablets EDTA-free, Roche). The suspension was transferred to a milliTUBE 1 ml AFA Fiber (part #520130) and processed in batch of 24 on an E220 focused-ultrasonicator from Covaris Inc. (Woburn, Massachusetts, USA) with the following settings: 20 min at 5% duty cycle, 140W, 200 cycles. The lysate was transferred to a 1.5 mL Eppendorf, centrifuged at 10,000g for 5 min to clear insoluble material – the supernatant was either frozen at -80°C or used right away for chromatin immunoprecipitation. Quality control was performed on a Bioanalyzer 2100 (Agilent) to verify that most fragments were between 200 and 600 bp. Chromatin immunoprecipitation was performed using anti-HA.11 antibody (clone 16B12, Covance), 15 µg per IP, and Protein G Dynabeads (ThermoFisher).
ChIP-exo was performed on bead-bound chromatin as described (Rhee and Pugh, 2011), with adaptations for use in 96-well plates and Illumina sequencing (detailed protocol available in Supplemental Information of the manuscript associated with this GEO entry).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw sequences were filtered according to the Purity flag using Illumina CASAVA-1.8 FASTQ Filter software before further processing.
Reads from each KRAB-ZNF library were mapped on GRCh37g1k (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/) using Bowtie2 (version 2.2.1) with the “very-sensitive-local” setting. Mapped reads were compressed in BAM files and indexed using SAMtools version 1.18.
We used MACS (version 1.4.2.1) with the “keep-dup all” parameter, which is recommended for ChIP-exo as genuine reads can accumulate on a few positions following exonuclease digestion. We devised a strategy based on random sampling of few reads from all ChIP-exo to build a composite total input, and verified that regions yielding very high signal for any one particular KRAB-ZNF were not enriched in our composite total input. An extensive filtering strategy was put in place as we found MACS to be prone to call peaks in various problematic regions of the human genome. Briefly, we filtered out peaks that would meet either of those criteria: MACS score < 80 (equivalent to a p-value of 1x10-8); within the blacklist of regions prone to false positives devised by ENCODE; ratio of forward vs. reverse strand reads greater than 4; less than 20 reads over 500 bp per 15 million reads; normalized read count was less than 2 fold over the control.
Genome_build: GRC h37 g1k
Supplementary_files_format_and_content: BED files from MACS after filtering to remove common artifacts
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| Submission date |
Jan 24, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Julien Duc |
| E-mail(s) |
julien.duc@epfl.ch
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| Organization name |
EPFL
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| Department |
School of Life Science
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| Lab |
LVG
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| Street address |
Station 19 CH-1015 Lausanne
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| City |
Lausanne |
| State/province |
VAUD |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE78099 |
ChIP-exo of human KRAB-ZNFs transduced in HEK 293T cells and KAP1 in hES H1 cells |
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| Relations |
| BioSample |
SAMN06252011 |
| SRA |
SRX2512818 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2466585_ZNF540_peaks_processed_score_signal_exo.bed.gz |
1.9 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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