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| Status |
Public on Jun 19, 2020 |
| Title |
Untreated_Pol2_Mitotic exit_Hi-C_rep1 |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Untreated Mitotic_mES
restriction enzyme: AluI
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| Treatment protocol |
All degron mES cells were pre-treated with 1 μg/ml Doxycycline for 12 hours. Pol II degron mES was treated with or without 500 μM indole-3-acetic acid (IAA) for 6 hours, whereas Pol I and Pol III degron mES were treated for 24 hours with same concentration. For the time course of Pol II degron, IAA was added 1 hour and 6 hours respectively. For synchronization Pol II degron mES cells were treated with 1.25 mM Thymidine for 14 hours, followed by treatment with 50 ng/ml Nocodazole for 7 hours; mitotic cells were collected at this time, mitotic exit cells were collected 1 hour after release. At the same time, all Pol II degron mES cells were pre-treated with 1ug/ml Doxycycline for 12 hours following being treated with or without 500 μM indole-3-acetic acid (IAA) for 6 hours.
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| Growth protocol |
The V6.5 mouse ES (mES) cell line was cultured with irradiated mouse embryonic fibroblasts in DMEM-KO containing 15% FBS, leukaemia inhibiting factor (LIF), L-glutamine, β-mercaptoethanol, non-essential amino acids and penicillin/ streptomycin at 37 ℃ with 5% CO2, and passage twice for experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Hi-C was conducted as BAT Hi-C (Huang et al., 2019). After AluI digestion, proximal ligation was mediated with biotin labeled linker, then remove unligated linker with ExoI and ligated DNA was extracted with streptavidin beads.
Llibrary was constructed with Illumina Nextera DNA Sample Preparation Kit (FC-121-1030), sequencing adaptor was added with Tn5 transposase.
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Description |
Pol2_untreated_Mitotic_downsampled_75mi_allValidPairs.hic
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| Data processing |
Linker detection: The bridge linker sequences were trimmed using trimlinker from ChIA-PET2 software, paired-end reads containing at least one instance of the bridge-linker in either end are adapted for further processing.
Reads alignment: The resulting reads were aligned against the mm10 genome and paired, filtered for long-range contacts, and removed of duplicates using the HiC-Pro pipeline (special paramters: --very-sensitive-local --local)
Filtering: Discard read-pairs marked as multihits, PCR duplicates, low mapping quality (MAPQ < 20), self-ligated fragments and dangling-end; only the valid read pairs involving two different restriction fragments were used to build the contact matrices.
Matrix generation: For unbiased comparison, we randomly sampled equal numbers of long range (>20 kb) intra-chromosomal read pairs (n=75 million) from each sample, Hi-C contact matrices with binned interactions (range from 5 kb to 100 kb) were generated.
Normalization: the raw Hi-C contact matrices were normalized using the iterative correction method with iced script to correct for systematic biases and the distance between restriction fragments as recently described.
Processed reads and downsampled allValidPairs were then inputted into the Juicebox pipeline (hicpro2juicebox.sh) to allow for visualization of the interaction matrix.
Genome_build: mm10
Supplementary_files_format_and_content: The processed files (.hic) were generated useing juicebox pre
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| Submission date |
Jun 19, 2020 |
| Last update date |
Jun 19, 2020 |
| Contact name |
Xiong Ji |
| E-mail(s) |
xiongji@pku.edu.cn
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| Organization name |
Peking University
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| Department |
School of Life Sciences
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| Lab |
Jilab
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| Street address |
Haidian District, Beijing Summer Palace Road No. 5
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (2) |
| GSE145873 |
Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II and Pol III [Hi-C] |
| GSE145874 |
Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II and Pol III |
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| Relations |
| BioSample |
SAMN15249329 |
| SRA |
SRX8562120 |
| 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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