|
| Status |
Public on Jul 19, 2017 |
| Title |
EGR1 ChIP Input Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
mesenchymal cells
|
| Organism |
Gallus gallus |
| Characteristics |
breed: Lohmann Selected Leghorn
tissue: limb buds
cell type: mesenchymal cells
developmental stage: E4.5
time of culture: 5 days
condition: RCAS-BP(A)-EGR1-3F
antibody: mouse anti-FLAG (Sigma-Aldrich, F1804)
|
| Treatment protocol |
Prior to seeding, mesenchymal cells were infected with RCAS-BP(A) retroviral particles carrying the CDS of connective tissue-asociated transcription factors.
|
| Growth protocol |
Mesenchymal cells were isolated from limb buds of E4.5 chick embryos and were plated as 10-µL droplets at a concentration of 2.10^7 cells/mL. Cells were maintained in culture for 5 days at 37°C in DMEM/Ham’s F-12 (1:1) medium (Biochrom) supplemented with 10% FBS (Biochrom), 0.2% chicken serum (Sigma-Aldrich), 1% L-glutamine (Lonza) and 1% penicillin/streptomycin (Lonza).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mesenchymal cells harvested from 24 cultures were cross-linked in 1% FA for 10 min on ice. Following cell lysis and nuclei isolation, nuclear extracts were sonicated to fragment chromatin between 200 and 500 bp. 30 µg of chromatin extracts were mixed with 10 µg of anti-FLAG (Sigma-Aldrich, F1804). Antibody-TF-DNA complexes were pulled down by using 40 µL of magnetic beads (Dynabeads protein G; Thermo Fischer). Enriched complexes were eluted from the beads. Reverse cross-linking was performed overnight at 65°C. DNA was purified by successive treatments with RNase A and Proteinase K followed by ethanol precipitation.
ChIP and input librairies were prepared by using the NEBNext Ultra DNA Library Preparation kit for Illumina (New England Biolabs). 50-bp single-end reads were generated by using a HiSeq 1500 sequencer (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
ChIP-seq single-end reads were filtered on their quality by using the FASTX-Toolkit (median quality value of minimum 28).
Filtered reads were mapped against the chicken genome galGal4 by using BWA (default parameters).
Uniquely mapped reads were extracted and duplicated reads were removed by using the tool rmdup from SAMtools.
Peak calling was performed as suggested by the ENCODE consortium by using MACS2 (--bw 130/135; -g 1.0e9; --to-large; -p 0.025).
Significant peaks were selected based on an IDR analysis (peak.half.width -1; min.overlap.ratio 0; is.broadpeak F; ranking.measure p.value) with a threshold fixed at 0.01.
Genome_build: galGal4
Supplementary_files_format_and_content: Peaks called by MACS2 in ENCODE narrowPeak format.
|
| |
|
| Submission date |
Jun 26, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Mickael Orgeur |
| Organization name |
Institut Pasteur
|
| Street address |
25-28 rue du Docteur Roux
|
| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
| |
|
| Platform ID |
GPL21476 |
| Series (2) |
| GSE100515 |
Genome-wide strategies identify downstream target genes of connective tissue-associated transcription factors [ChIP-seq TFs] |
| GSE100517 |
Genome-wide strategies identify downstream target genes of connective tissue-associated transcription factors |
|
| Relations |
| BioSample |
SAMN07280070 |
| SRA |
SRX2959157 |
