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| Status |
Public on Jun 17, 2024 |
| Title |
IMR90, ICM, replicate 2, eclip |
| Sample type |
SRA |
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| Source name |
IMR90
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| Organism |
Homo sapiens |
| Characteristics |
eclip antibody: CTCF(Active Motif 61331)
cell line: IMR90
cell type: primary lung fibroblasts
genotype: WT
treatment: ICM inducing senescence
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| Growth protocol |
Proliferating primary lung fibroblasts (IMR90) isolates (I79 and I83, passage 5; Coriell) were grown in MEM (M4655, Sigma-Aldrich) supplemented with 1x non-essential amino acids and 10% FBS under 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
~20x10^6 IMR90 were UV-crosslinked (400 mJ/cm2 constant energy), lysed in iCLIP lysis buffer, and sonicated (on a BioRuptor Pico). Lysates were treated with RNase I (Ambion, AM2294) to fragment RNA, after which CTCF protein-RNA complexes were immunoprecipitated using the relevant antibody (anti-CTCF, Active Motif 61331). In parallel to the IP, an “input” library was generated for each replicate for which no antibody was used. Stringent washes were next performed, during which RNA was dephosphorylated with FastAP enzyme (Fermentas) and T4 PNK (NEB, M0201S). Subsequently, a 3’ RNA adaptor oligonucleotide was ligated onto the RNA using a T4 RNA ligase (NEB, M0242S). Protein-RNA complexes were separated on an SDS-polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes, and RNA was isolated off the membrane. After precipitation, RNA was reverse-transcribed with AffinityScript reverse transcriptase (Agilent, 600107), free oligos were removed using ExoSap-IT (Thermo Fisher Scientific, 78201.1.ML), and a 3’ DNA adaptor was ligated onto the cDNA product.
Libraries were then amplified with 2x Q5 PCR mix (NEB)
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
*library strategy: eCLIP
The data were analyzed via the eCLIP pipeline (https://github.com/YeoLab/eCLIP)
Assembly: hg38
Supplementary files format and content: bigWig separated by strandness
Supplementary files format and content: narrowPeaks
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| Submission date |
Jun 14, 2024 |
| Last update date |
Jun 17, 2024 |
| Contact name |
Argyris Papantonis |
| E-mail(s) |
argyris.papantonis@med.uni-goettingen.de
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| Organization name |
University Medical Center Göttingen
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| Department |
Institute of Pathology
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| Street address |
Robert-Koch-Str. 40
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| City |
Göttingen |
| ZIP/Postal code |
37075 |
| Country |
Germany |
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| Platform ID |
GPL21290 |
| Series (1) |
| GSE269857 |
Repurposed nuclear speckle and mitosis components induce CTCF clustering to sustain the senescence splicing program [eCLIP] |
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| Relations |
| BioSample |
SAMN41834488 |
| SRA |
SRX24925899 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8329000_ICM_rep2.IP.umi.r1.fq.genome-mappedSoSo.rmDupSo.norm.neg.bw |
4.3 Mb |
(ftp)(http) |
BW |
| GSM8329000_ICM_rep2.IP.umi.r1.fq.genome-mappedSoSo.rmDupSo.norm.pos.bw |
4.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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