NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM5387134 Query DataSets for GSM5387134
Status Public on Sep 06, 2022
Title Input_Runx2-FLAG_Cho_1
Sample type SRA
 
Source name P1 rib chondrocytes
Organism Mus musculus
Characteristics cell type: Rib chondrocytes at P1
strain: C57BL/6
genotype: Runx2-BioFL/+ knock-in
Treatment protocol For in vitro cell culture, osteogenic medium contains 10% FBS/DMEM supplemented with 50 μg/ml ascorbic acid phosphate, 10 mM β-glycerophosphate, and 100 ng/ml rhBMP‑2
Extracted molecule genomic DNA
Extraction protocol Rib chondrocytes were manually dissected from the neonatal mouse ribs. The ribs were initially digested in 2 mg/mL Pronase (53702, EMD Millipore Corporation) for 30 min at 37 °C, washed three times with phosphate buffered saline (PBS), and then digested in 0.2% collagenase D (1088858, Roche Applied Science) for 3 h at 37 °C. Calvarial osteoblasts were manually dissected from osteoblast-enriched partial calvarias consisting of the frontal bone and a rostral part of the parietal bone. The partial calvarias were digested in five changes of 0.1% collagenase D and 0.2% dispase for 10 min each at 37 °C. Cultured 3T3 cells were digested in trypsin-EDTA solution 1X (Wako, 206-17291) for 5 to 15 min at 37 °C.
ChIP-seq libraries were constructed using a ThruPLEX®-FD Prep Kit (R40012, Rubicon Genomics) according to the manufacturer′s instructions. ATAC-seq libraries were constructed as previously described (Buenrostro et al., 2013).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer II
 
Data processing DNA-sequence information was aligned to the unmasked mouse genome by bowtie
Peak calling was performed by two-sample analysis on CisGenome software (Ji et al., 2008) with a P-value cutoff of 10-5 comparing with the negative control
In the analysis with ATAC_Col2, ATAC_Col10 and ATAC_Sp7 data sets, to identify the cell-type specific and shared regions, we selected ATAC-seq peaks with the ATAC-seq normalized peak intensity based on the following criteria: raw fe > 30, fold change > 5, and p-value < 0.05, for cell-type specific regions. The differential accessible regions were called as below. C2: C2-Cho-distinct peaks; C10: C10-Cho-distinct peaks; S7: S7-Ob-distinct peaks; C2C10: peaks with high intensity in both C2-Cho and C10-Cho but not in S7-Ob; C10S7: peaks with high intensity in both C10-Cho and S7-Ob but not in C2-Cho; C2S7: peaks with high intensity in both C2-Cho and S7-Ob but not in C10-Cho. These peaks were listed in "ATAC_C2_C10_Sp7_peaks.xlsx”
In the analysis with ATAC_POB_Runx2_cHetKO and ATAC_POB_Runx2_cHomKO data sets, to identify the differential binding regions between the genotypes, DiffBind was applied with the following parameters "summits = 150" in the step of "dba.count" to resize peaks. To obtain statistically different chromatin-accessible regions, we selected the peaks based on FDR < 0.05. The differential accessible regions were listed in “ATAC_Runx2KO_peaks. xlsx”
In the analysis with ATAC_3T3cells data sets, to identify Runx2-responsive and osteoblast induction-responsive regions, DiffBind was applied with the following parameters "summits = 250" in the step of "dba.count" to resize peaks. Pairwise comparison was performed among ATAC-seq profiles of day 0 GFP expression samples, day 0 Runx2 expression samples, day 3 GFP expression samples, day 3 Runx2 expression samples, and no expression samples (None). The Runx2-responsive and the osteoblast induction-responsive regions were selected as ATAC-seq peaks whose intensity was statistically upregulated by both Runx2 expression and osteogenic induction on day 3, based on the following criteria: FDR < 0.05, fold change > 4. The Runx2-responsive regions were listed in “ATAC_3T3_Runx2_responsive_regions.xlsx”.
Genome_build: mm9
Supplementary_files_format_and_content: peak files
 
Submission date Jun 16, 2021
Last update date Sep 06, 2022
Contact name Hironori Hojo
E-mail(s) hojo@tetrapod.t.u-tokyo.ac.jp
Phone +81-3-5841-1427
Organization name The University of Tokyo
Department Center for Disease Biology and Integrative Medicine
Lab Clinical Biotechnology
Street address 7-3-1 Hongo
City Bunkyo-ku
State/province Tokyo
ZIP/Postal code 113-8655
Country Japan
 
Platform ID GPL9250
Series (2)
GSE178291 Roles of Runx2 in chromatin landscape in skeletal development [ChIP-Seq, ATAC-Seq]
GSE178293 Roles of Runx2 in chromatin landscape in skeletal development
Relations
BioSample SAMN19727843
SRA SRX11156881

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not applicable for this record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap