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| Status |
Public on Dec 07, 2020 |
| Title |
LSC_shRUNX1_H3K27AC_rep2 |
| Sample type |
SRA |
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| Source name |
limbal stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: limbal stem cells
treatment: shRUNX1 treated
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| Treatment protocol |
For ChIP-seq, chromatin was fixed with 1% formaldehyde for 10 min and sheared in a sonification buffer. Fragmented DNA was incubated with primary antibodies overnight at 4℃ and then with Protein A/G Dynabeads for 1 h. The beads were collected and washed sequentially with the following solutions: high-salt buffer, low-salt wash buffer, and TE buffer. Elution of the immunoprecipitated protein/DNA complexes was performed using elution buffer at 65℃ for 4 h. Next, the protein/DNA complexes were incubated with proteinase K (Invitrogen) and RNase A (Invitrogen) for 1 h. De-crosslinked DNA was purified using the PCR Purification Kit (Qiagen). For ATAC-seq, In total, 50,000 cells were digested, collected, and lysed in ice-cold lysis buffer for 5 min. Then, Tn5 transposase reactions (Vazyme Biotech, TD501) were performed using the TruePrep DNA Library Prep Kit (Vazyme Biotech).
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| Growth protocol |
LSCs and SESCs were grown in 1:1 DMEM/F-12 medium containing 1% penicillin/streptomycin, 10% fetal bovine serum, 10 ng/ml EGF, 5 μg/ml insulin, 0.4 μg/ml hydrocortisone, 0.1 nM cholera toxin and 2 nM 3,3',5-triiodo-L-thyronine.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Purified DNA was used to prepare ChIP-seq DNA libraries for Illumina sequencing by KAPA Hyper Prep Kit (KAPABIOSYSTEMS). For ATAC-seq library, the transposed DNA fragments were amplified and collected using the TruePrep DNA Library Prep Kit (Vazyme Biotech).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
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| Data processing |
Reads were trimmed by trimmomatic tool and then aligned to human hg19 reference genome using BWA software (version 0.7.17).
Duplicated reads were removed by Picard Markduplicates (version 2.18.16) and only uniquely mapping reads were retained for further analysis.
MACS2 (version 2.1.1) was used to call peak. For sharp peaks, we used the following parameters: -f BAMPE -B --SPMR -q 0.001 --call-summits --fix-bimodal --seed 11521 --extsize 200. For broad peaks (H3K27me3 and H3K4me1), the following parameters were used: -f BAMPE -B --SPMR --fix-bimodal --extsize 500 --broad --broad-cutoff 0.1 --seed 11521.
Genome_build: hg19
Supplementary_files_format_and_content: Peak files with enriched genomic site and quantitative value.
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| Submission date |
Aug 14, 2020 |
| Last update date |
Dec 07, 2020 |
| Contact name |
Mingsen Li |
| E-mail(s) |
lims3@mail2.sysu.edu.cn
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| Organization name |
Sun Yat-sen University
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| Department |
Zhongshan Ophthalmic Center
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| Street address |
No. 7 Jinsui road Tianhe district
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| City |
Guanzhou |
| ZIP/Postal code |
510060 |
| Country |
China |
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| Platform ID |
GPL20795 |
| Series (2) |
| GSE156272 |
Core transcription regulatory circuitry orchestrates stratified epithelial homeostasis (ChIP-seq, ATAC-seq) |
| GSE156273 |
Core transcription regulatory circuitry orchestrates stratified epithelial homeostasis |
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| Relations |
| BioSample |
SAMN15822655 |
| SRA |
SRX8948973 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4728074_shRUNX1_H3K27AC_rep2_call_peak.xlsx |
2.3 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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