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| Status |
Public on Feb 17, 2015 |
| Title |
ChIP-seq of GFP-tagged ZNF45 in HEK293 cells |
| Sample type |
SRA |
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| Source name |
HEK293 cells expressing GFP-tagged protein
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| Organism |
Homo sapiens |
| Characteristics |
host cell line: HEK293
transfected with: GFP-tagged ZNF45
uniprotkb id: Q02386
chip antibody: polyclonal anti-GFP antibody
chip antibody vendor: Abcam
chip antibody cat. #: ab290
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| Growth protocol |
HEK293 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics. Gateway-compatible entry clones were cloned into the pDEST pcDNA5/FRT/TO-eGFP vector according to the manufacturer's instructions and co-transfected into Flp-In T-REx 293 cells together with the pOG44 Flp recombinase expression plasmid. Cells were selected for FRT site-specific recombination into the genome, following instructions by the manufacturer. Expression of the gene of interest was induced by addition of doxycycline to the culture medium 24 hours prior to harvesting.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HEK293 cells were cross-linked for 10 min in 1% formaldehyde. Lysates were sonicated to a DNA fragment length range of 200-300 bp using a Bioruptor (Diagenode). GFP-tagged transcription factors were immunoprecipitated with a polyclonal anti-GFP antibody (ab290, Abcam) and Dynabeads Protein G (Invitrogen). Subsequently, crosslinks were reversed at 65°C over night and bound DNA fragments were purified (EZ-10 Spin Column PCR Product Purification kit, Bio Basic).
Sequencing libraries were constructed using the NEBNext ChIP-Seq Library Prep kit (NEB) according the manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
ENSG00000124459
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| Data processing |
The 3’ end of reads were trimmed so that the final reads were 50 nucleotides long, and the reads were mapped to the human genome build GRCh37 using Bowtie 2 (29), with “--very-sensitive” preset of parameters. This parameter set allows one alignment to be reported for each read that is mapped to multiple positions on the genome, in addition to alignments of uniquely mappable reads. Duplicate reads, which often represent PCR amplification artifacts, were removed using SAMtools.
We performed two rounds of peak-calling: an initial round was performed without an explicit background model of read distributions, in order to identify ChIP experiments with similar experimental/read-mapping biases. We then constructed a background model for each sample, and repeated the peak-calling in order to identify the final set of protein-specific peaks. For the initial round, peaks were identified using MACS v1.4, with fragment length specified based on the results of cross-correlation analysis using SPP package (average 150 bp). Then, the overlap of the identified peaks for each pair of experiments was calculated as the total length of the intersection of peaks divided by the total length of the union of peaks from two experiments. Then, for each experiment x, the top 10 experiments with the highest overlap were identified, so that none of these 10 experiments would represent a biological replicate of experiment x. The reads from these 10 experiments were then randomly sampled, with probabilities proportional to the extent of overlap of each experiment with experiment x, so that a total of 5e+7 reads would be obtained. These reads were then used as the background model in order to perform a second, final round of peak-calling for experiment x, using MACS.
Genome_build: hg19
Supplementary_files_format_and_content: BED files containing peaks and peak summits
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| Submission date |
Jun 10, 2014 |
| Last update date |
Oct 24, 2024 |
| Contact name |
Hamed S Najafabadi |
| Organization name |
McGill University
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| Department |
Human Genetics
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| Lab |
Computational and Statistical Genomics Lab
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| Street address |
740 Dr. Penfield Avenue, Room 7202
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| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H3A 0G1 |
| Country |
Canada |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE52523 |
Large-scale characterization of DNA binding landscape of eukaryotic C2H2 zinc fingers |
| GSE58341 |
Identification of in vivo binding sites of human C2H2-ZF proteins |
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| Relations |
| BioSample |
SAMN02848825 |
| SRA |
SRX585473 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1407651_ENSG00000124459_peaks.bed.gz |
75.7 Kb |
(ftp)(http) |
BED |
| GSM1407651_ENSG00000124459_summits.bed.gz |
57.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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