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| Status |
Public on Sep 06, 2022 |
| Title |
Input_Runx2-FLAG_Cho_1 |
| Sample type |
SRA |
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| Source name |
P1 rib chondrocytes
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| Organism |
Mus musculus |
| Characteristics |
cell type: Rib chondrocytes at P1
strain: C57BL/6
genotype: Runx2-BioFL/+ knock-in
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| 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
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| 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).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| 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
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| 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
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| Phone |
+81-3-5841-1427
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| Organization name |
The University of Tokyo
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| Department |
Center for Disease Biology and Integrative Medicine
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| Lab |
Clinical Biotechnology
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| Street address |
7-3-1 Hongo
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| City |
Bunkyo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
113-8655 |
| Country |
Japan |
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| 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 |
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| Relations |
| BioSample |
SAMN19727843 |
| SRA |
SRX11156881 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not applicable for this record |
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