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| Status |
Public on Mar 26, 2024 |
| Title |
22Rv1-WT-Pol2s5-ChIP-seq |
| Sample type |
SRA |
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| Source name |
22Rv1_Cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: 22Rv1
genotype: 22Rv1-WT
antibody: Pol2s5 (Abcam, ab5131, lot: GR3238867-3)
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| Treatment protocol |
HKI cell lines were acquired by Crispr/Cas9 mediated HOTAIR poly-A knock in .
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| Growth protocol |
22Rv1 cell line was cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA-seq:Total RNA was extracted using TRIzol according to the manufacturer’s instruction. Then, ribosomal RNA was deleted from 5 μg of total extracted RNA using the Ribo-Zero Gold Kit, using Illumina novaseq 6000. ChIRP-seq: Cells were fixed with glutaraldehyde for 10 min and nucleus was isolated and sheared to 100-500 bp fragments by sonication, then hybridized with DNA probes. Libraries were prepared using the NEBNext DNA library kit according to the manufacturer's instructions (New England BioLabs), using Illumina novaseq 6000. ChIP-sq:Cells were fixed with formaldehyde for 10 min and nucleus was isolated and sheared to 200-500 bp DNA fragments by sonication, then immunoprecipitated with the corresponding antibodies. Libraries were prepared using the NEBNext DNA library kit according to the manufacturer's instructions (New England BioLabs), using Illumina novaseq 6000. Cut and tag: The experiments were performed using Hyperactive Universal CUT & Tag Assay Kit (Vazyme, TD901). Briefly, Cells were incubated with Concanavalin A- Coated beads and then incubated with antibody. DNA sequencing library was built by pA-Tn5 transpose, then sequenced by Illumina novaseq 6000.
RNA-seq, ChIRP-seq , ChIP-seq and Cut and tag libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Clean reeas of RNA-seq were mapped to hg38 using STAR.RNA-seq datasets were performed using the TOPMed RNA-seq pipeline . Differentially expressed genes were identified using DESeq2 according to default parameters .Genes with an absolute log2 fold change > 1 and an adjusted P-value < 0.05 were considered as differentially expressed ones
Clean reads of ChIRP-seq were mapped to hg38 using STAR. Normalized bigwigs of even, odd, and merged files were generated using the ChIRP-seq processing scripts . Peaks of merged bigwig were called using MACS2, and called peaks were filtered to exclude blacklist regions from ENCODE. Peaks with correlation of even and odd > 0.8, FDR < 0.05, and read coverage > 2-fold of the input were kept as confident HOTAIR target regions.
Clean reads of ChIP-seq and Cut and tag were mapped to hg38 using STAR.Biological replicates that showed a good correlation (Pearson correlation coefficient > 0.8) were merged for downstream analysis, including peak calling and plots. Peaks for histone modifications were called using MACS2 , peaks for transcription factors were called using SPP.Genomic annotation for called peaks was performed using Homer with default parameters. Normalized coverage track (bigWig) files were generated using bamCoverage from deepTools.
Assembly: hg38
Supplementary files format and content: xlsx file include genes counts values and bw files include ChIP, ChIRP, Cut and tag signal peaks for each Sample
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| Submission date |
Dec 19, 2022 |
| Last update date |
Mar 26, 2024 |
| Contact name |
Yuyang Qian |
| E-mail(s) |
qyy@nankai.edu.cn
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| Organization name |
Nankai university
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| Department |
cell and genetic department
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| Street address |
94 Weijin Road
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| City |
Tianjin |
| State/province |
Tianjin |
| ZIP/Postal code |
300071 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE221263 |
HOTAIR promotes prostate cancer progression by mediating the interaction between Pol2S2 and CDK9 to maintain transcriptional elongation |
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| Relations |
| BioSample |
SAMN32340937 |
| SRA |
SRX18799695 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6857384_Pol2s5-22Rv1-WT-ChIP-seq.nodup.bw |
53.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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