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Status |
Public on Jun 07, 2023 |
Title |
2.Hnf4a2Tg.av.rep2 |
Sample type |
SRA |
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Source name |
Calvaria
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Organism |
Mus musculus |
Characteristics |
tissue: osteoblast-like age: 21 days of culture genotype: HNF4a2Tg cell line: MC3T3-E1 subclone 4 (ATCC CRL-2593) expression vector: HNF4a2 cDNA chip antibody: OASG03561 (Aviva Systems Biology, San Diego, CA, USA)
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Growth protocol |
Cells were cultured for 21 days in αMEM medium supplemented with 10% FBS (Corning, New York, USA), 10 U/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific, Whaltman, USA), 10 mM β-glycerophosphate, and 50 μg/ml ascorbic acid (Sigma-Aldrich, St. Louis, USA) to induce differentiation.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cell cultures were subjected to ChIP assay following the protocol provided by manufacturer (SimpleChIP Plus Enzymatic Chromatin IP Kit with magnetic beads, Cell Signaling Technology, Danvers MA, USA). Briefly, protein-chromatin crosslinking was carried out in cell medium containing 1% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 10 minutes. After stopping the crosslinking reaction using 10X glycine for 5 min, cells were washed 3 times in PBS and scraped into cold PBS containing protease inhibitor cocktail. Collected cells were centrifuged at 2000xg for 5 min at 4C. After nuclei extraction, chromatin was digested with micrococcal nuclease, and nuclear membranes were disrupted by sonication. Lysates were clarified by centrifugation at 9400xg for 10 min at 4C. Per IP reaction, 10 µg of digested, cross-linked chromatin were incubated with anti-HNF4α antibodies for immunoprecipitation: 5 µg of OASG03561 (Aviva Systems Biology, San Diego, CA, USA), 10 µg of ab41898 (Abcam, Cambridge, UK), or 10 µg of anti-Halotag antibody (G9281, Promega, Madison, WI, USA) for 4h at 4C. Then, 30 μl of Dynabeads Protein G Magnetic Beads was added to the IP chromatin solution and incubated for 2h at 4C. After several washing steps using buffers with ascending NaCl concentrations, chromatin was eluted, and the supernatant was incubated with proteinase K overnight at 65C to reverse crosslinking. Finally, DNA fragments were purified using silica columns. The total DNA library for each individual sample was prepared using the TruSeq ChIP-Seq Library Prep Kit (Illumina, San Diego, CA), and the bar-coded cDNA libraries were sequenced for 100 bp single reads using the Illumina Hiseq 4000. The ENCODE pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline2) v 1.7.1 was used to identify naive overlapping peaks in each experiment. Enriched regions were consolidated based on their representation in two or more experiments as follow: HNF4α1/2 peaks if detected in samples expressing Hnf4α1 and Hnf4α2; HNF4α1 peaks if detected in two or more samples overexpressing Hnf4α1 or Ctr cells; HNF4α2 peaks if detected in two or more samples overexpressing Hnf4α2 or Ctr cells.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 4000 |
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Data processing |
The Encode Chipseq pipeline2 v 7.0 (https://github.com/ENCODE-DCC/chip-seq-pipeline2) was used to process the samples from fastqs to overlapping naïve peaks. Alignment:bwa Genome_build: mm10 Peak caller:spp with fdr 0.01 Supplementary_files_format_and_content: *peak.regionPeak.gz: encode region peak files for each individual experiement bed file for overlapping peaks from separate experiments HNF4a1_Peaks.bed: Peaks found in 2 or more experiments for which in at least one experiment cells overexpress Hnf4a1. The other(s) peaks should derive from either Ctr cells and/or cells overexpressing Hnf4a1. HNF4a2_Peaks.bed: Peaks found in 2 or more experiments for which in at least one experiment cells overexpress Hnf4a2. The other(s) peaks should derive from either Ctr cells and/or cells overexpressing Hnf4a2. HNF4a1-2_Peaks.bed: Peaks found in 2 or more experiments for which in at least one experiment cells overexpress Hnf4a1 and at least one Hnf4a2. The other(s) peaks should derive from either Ctr cells and/or cells overexpressing Hnf4a1 or Hnf4a2.
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Submission date |
Dec 06, 2021 |
Last update date |
Jun 07, 2023 |
Contact name |
Valentin David |
E-mail(s) |
valentin.david@northwestern.edu
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Phone |
13125034159
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Organization name |
Northwestern University
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Department |
Medicine
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Lab |
David Lab
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Street address |
320 E Superior Street
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City |
Chicago |
State/province |
Illinois |
ZIP/Postal code |
60611 |
Country |
USA |
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Platform ID |
GPL21103 |
Series (1) |
GSE190314 |
Role of HNF4a in osteoblasts [ChIP-seq] |
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Relations |
BioSample |
SAMN23708491 |
SRA |
SRX13331623 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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