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| Status |
Public on Nov 06, 2019 |
| Title |
ChIPseq_IMR90_CAPH2_FLAG_OIS2 |
| Sample type |
SRA |
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| Source name |
IMR90 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90
cell type: Lung fibroblasts
genotype/variation: wild type
cell status: OIS (Oncogene-Induced Senescence) cells
passage: 28-36
karyotype: Normal diploid
chip_antibody: Mouse monoclonal anti-FLAG (clone M2, Sigma Aldrich),
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| Treatment protocol |
pBABE-puro plasmids carrying H-rasV12 were used for retrovirus packaging, and virus infection was performed. H-rasV12-expressing cells were cultured in medium containing 1 µg/ml puromycin for 7 days to obtain OIS cells. pTRIPZ plasmids containing NCAPH2 shRNAs (#1, V3THS_326285; #2, V3THS_326286; #3, V3THS_326290, Dharmacon) were used for CAP-H2 knockdown. Lentivirus was prepared using ViraPower kit (Thermo Fisher Scientifc) according to the manufacturer’s protocol. Doxycycline (1μg/ml) was added to culture medium every 48 hours after lentiviral transfection for shRNA expression. OIS cells were further infected with lentivirus encoding one of the three shRNA constructs (#1, #2, and #3) against NCAPH2 and harvested 3 days after the lentivirus infection.
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| Growth protocol |
Normal diploid IMR90 human fibroblasts were cultured in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.15% sodium bicarbonate, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1 ´ MEM non-essential amino acids. BJ human fibroblasts were cultured in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
In situ Hi-C was performed as previously described (Rao et al., 2014, Cell). ChIP-seq was performed as previously described (Aird et al., 2017 JCB). RNA was extracted from IMR90 cells using RNeasy Mini kit (Qiagen). RNA-seq was carried out as previously described with modifications (Tanizawa et al. 2017 NSMB).
For in situ Hi-C, sequencing adapters were ligated using NEBNext Ultra Ligation module (New England Biolabs, Inc) on Dynabeads, and DNA was PCR-amplified for 8-14 cycles using NEBNext Q5 Hot Start HiFi PCR master Mix and NEBNext multiplex oligos (New England Biolabs, Inc). ChIP-seq was carried out as described previously (Iwasaki et al. 2015). Approximately 1 ng ChIP DNA was subjected to the illumine library construction using NEBNext Ultra DNA library prep kit (New England Biolabs). Adaptor-ligated DNA was PCR-amplified for 12 cycles using NEBNext Q5 Hot Start HiFi PCR master Mix and NEBNext multiplex oligos (New England Biolabs). PCR products were sequenced on Illumina NextSeq 500 platform to obtain 76-bp single-end reads. For RNA-seq, Poly (A) RNA was purified using NEBNext Poly (A) mRNA Magnetic Isolation Module (New England Biolabs), and sequence libraries were constructed using NEBNext mRNA Library prep Master mix for Illumina (New England Biolabs). Adaptor-ligated DNAs were PCR-amplified for 12 cycles using NEBNext Q5 Hot Start HiFi PCR master Mix and NEBNext multiplex oligos (New England Biolabs). PCR products were sequenced on Illumina NextSeq 500 platform to obtain 76-bp single-end reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Validated by RNAi & western blot
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| Data processing |
ChIP-seq: 76-bp or 50-bp reads were aligned to the human genome (hg19) using Bowtie2 (version 2.2.9).
ChIP-seq: Redundant reads were removed by picard (version 2.7.1). Low quality read (MapQ < 10) were also removed.
ChIP-seq peaks were defined by HOMER software (version 4.8.3) using the option “-center -style factor -F 1 -P 0.0001 -fdr 0.05” for RNA Pol II and TP53, while the alternative option “-center -style histone -F 2 -P 0.0001 -fdr 0.05” was used for other proteins. Input samples were used for the control of peak calling.
ChIP-seq score of more than 7-fold compared to the Input were removed from the bigwig file.
Supplementary_files_format_and_content: ChIP-seq: bigwig file were generated using bedGraphToBigWig (version 4)
RNA-seq: Sequenced reads were aligned to the human genome (hg19) using the STAR program (v2.5.2)
RNA-seq: Reads assigned to exons were estimated by the RSEM program (v1.2.31) ), and genes carrying less than 10 reads were eliminated, resulting in total 19,591 genes.
RNA-seq: Read numbers in different samples were normalized using the DEseq2 program ver3.7
RNA-seq: The DEseq2 program was used to identify differently expressed genes. CAP-H2 KD samples (KD#1 and KD#3) were treated as biological replicas. FDR < 0.05 was used as a threshold to identify significantly up- or down-regulated genes.
Supplementary_files_format_and_content: ChIP-seq: bigwig file were generated using bedGraphToBigWig (version 4)
Supplementary_files_format_and_content: RNA-seq: tab-deliminated text files of ensemble gene ID and expected read count obtained from RSEM calculation.
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| Submission date |
Sep 05, 2018 |
| Last update date |
Nov 07, 2019 |
| Contact name |
Hideki Tanizawa |
| E-mail(s) |
hidekit@uoregon.edu
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| Organization name |
University of Oregon
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| Department |
Institute of Molecular Biology
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| Street address |
1370 Franklin Blvd
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| City |
Eugene |
| State/province |
OR |
| ZIP/Postal code |
97403 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE118494 |
Involvement of Condensin in Cellular Senescence through Gene Regulation and Compartmental Reorganization |
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| Relations |
| BioSample |
SAMN09976692 |
| SRA |
SRX4643604 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3375538_ChIPseq_IMR90_CAPH2_FLAG_OIS2.bw |
139.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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