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Sample GSM5719693

Query DataSets for GSM5719693

Status

Public on Jun 07, 2023

Title

6.Hnf4a1Tg.ab.rep2

Sample type

SRA

Source name

Calvaria

Organism

Mus musculus

Characteristics

tissue: osteoblast-like
age: 21 days of culture
genotype: HNF4a1Tg
cell line: MC3T3-E1 subclone 4 (ATCC CRL-2593)
expression vector: HNF4a1 cDNA
chip antibody: ab41898 (Abcam, Cambridge, UK)

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.

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.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

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.

Submission date

Dec 06, 2021

Last update date

Jun 07, 2023

Contact name

Valentin David

E-mail(s)

valentin.david@northwestern.edu

Phone

13125034159

Organization name

Northwestern University