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| Status |
Public on Oct 10, 2024 |
| Title |
mCherry-IGF2BP1 Y396E, control, rep1, RIP |
| Sample type |
SRA |
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| Source name |
HCT116
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
cell type: human colorectal carcinoma
genotype: mCherry-IGF2BP1 Y396E, IGF2BP2 KO, IGF2BP3 KO, Tet-OsTIR1-PURO
treatment: PBS control
sample type: RIP-Seq
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| Treatment protocol |
To induce stress cells were treated with 500 µM sodium arsenite (Sigma) for two hours before collection. PBS was added to the control cells.
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| Growth protocol |
HCT116 conditionally expressing Tet-OsTIR1 were obtained from Masato Kanemaki Lab and cultured in McCoy's 5A (Modified) Medium (Sigma) with 10% Fetal Bovine Serum (Gibco), 2 mM Glutamine (Sigma), 1% Pen/Step (Sigma).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed with ice-cold PBS, scraped, centrifuged (3500 rpm, 4 min) and the pellet flash frozen and stored at -80°C. On the day of the experiment, the pellets were thawed and 150 µL of lysis buffer (25 mM HEPES pH 7.3, 150 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT). 1x protease inhibitor cocktail (Roche), 1x phosphatase inhibitor PhosSTOP (Roche) and 1 U / µL of Murine RNase Inhibitor (New England Biolabs) were added for lysis. Cells were lysed by vortexing for 20 minutes (3 seconds with 2-minute intervals). The lysate was centrifuged at 20.000 rpm for 15 minutes. 20 µL of RPF-Trap magnetic agarose (rtma) were used per 10 cm dish. Lysates were incubated at 4°C for 1 h rotating, and then washed three times with lysis buffer and twice with wash buffer (25mM HEPES pH 7.3, 150mM NaCl, 1mM MgCl2, 0.5% NP40). Then the beads were resuspended in 150 µL of wash buffer containing 0.1% SDS and 2 mg/mL proteinase K (Ambion), and incubated at 55°C for 30min. The eluate was collected and purified with an RNA cleanup kit (Zymo Research). The purified RNA samples were rRNA depleted using human riboPOOL probes (siTOOLs) according to the manufacturer’s instructions. The RNA was then purified with an RNA cleanup kit (Zymo Research) and treated with 0.2 U of RNase-free DNase I (NEB) at 37°C for 15 min, and re-purified with an RNA cleanup kit (Zymo Research)
Libraries were prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB).
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq X |
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| Description |
Y396E_C_1_RIP
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| Data processing |
The RIP-Seq libraries were sequenced on NovaSeqX 1.5B at SingleRead 100 mode at the Vienna BioCenter NGS facility producing ~35 million reads per sample. BCL files were converted to demultiplexed fastq files with bcl2fastq v2.20.0.422. The quality of fastq files was checked with fastqc 0.11.9.
The fastq files were trimmed and aligned to human genome hg38 using GENCODE annotation (release 46) with STAR v2.7.11b allowing for the two mismatches. Gene counts were obtained using FeatureCounts function of the Subread package v2.0.6.
Assembly: hg38, GENCODE annotation (release 46)
Supplementary files format and content: tsv, gene read counts
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| Submission date |
Jul 23, 2024 |
| Last update date |
Oct 10, 2024 |
| Contact name |
Aleksandra S Anisimova |
| E-mail(s) |
aleksandra.anisimova@univie.ac.at, alessandrick@gmail.com
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| Organization name |
Max Perutz Labs, Vienna BioCenter, University of Vienna, Medical University of Vienna
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| Lab |
Karagöz Lab
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| Street address |
Dr. Bohr Gasse 9
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL34281 |
| Series (2) |
| GSE272873 |
IGF2BP1 phosphorylation regulates ribonucleoprotein condensate formation by impairing low-affinity protein and RNA interactions (RIP-Seq) |
| GSE272875 |
IGF2BP1 phosphorylation regulates ribonucleoprotein condensate formation by impairing low-affinity protein and RNA interactions |
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| Relations |
| BioSample |
SAMN42763159 |
| SRA |
SRX25423408 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8413897_Y396E_C_1_RIP.tsv.gz |
3.8 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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