|
| Status |
Public on Oct 25, 2016 |
| Title |
ChIP-seq of GFP-tagged ZNF770 in HEK293 cells [ZNF770_rep1] |
| Sample type |
SRA |
| |
|
| Source name |
HEK293 cells expressing GFP-tagged protein
|
| Organism |
Homo sapiens |
| Characteristics |
uniprotkb id: Q6IQ21
cell line: HEK293
|
| Treatment protocol |
Expression of the gene of interest was induced by addition of doxycycline to the culture medium 24 hours prior to harvesting. HEK293 cells were cross-linked for 10 min in 1% formaldehyde.
|
| 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.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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 TruSeq ChIP-seq library kits (Illumina) according the manufacturer's instructions followed by PCR amplification and agarose gel size selection.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
ChIP-seq reads were mapped to the human genome build hg19 using Bowtie 2. The 3’ end of reads were trimmed to a final length of 50 nucleotides, mapped with the Bowtie “--very-sensitive” preset of parameters, and duplicate reads were removed using SAMtools.
MACS v1.4 was used for peak calling, using experiment-specific background models constructed by pooling multiple control datasets.
Genome_build: hg19
Supplementary_files_format_and_content: MACS v1.4 output, containing the coordinates of peaks and summits based on hg19 genome assembly, as well as associated scores.
Supplementary_files_format_and_content: combined_summits.per_protein.hg19.tar: Tar of peak summits per protein.
Supplementary_files_format_and_content: combined_summits.motif_hits.per_protein.hg19.tar: Tar of motif hit locations within peaks per protein.
Supplementary_files_format_and_content: motifs.pfm.txt: Tab-delimited text file includes motifs identified for each protein.
|
| |
|
| Submission date |
Jan 04, 2016 |
| Last update date |
Oct 24, 2024 |
| Contact name |
Hamed S Najafabadi |
| Organization name |
McGill University
|
| Department |
Human Genetics
|
| Lab |
Computational and Statistical Genomics Lab
|
| Street address |
740 Dr. Penfield Avenue, Room 7202
|
| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H3A 0G1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE76494 |
Identification of in vivo binding sites of human GFP-tagged C2H2-ZF proteins |
| GSE76496 |
Multiparameter functional diversity of human C2H2 zinc finger proteins |
|
| Relations |
| BioSample |
SAMN04383239 |
| SRA |
SRX1514992 |
