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| Status |
Public on Aug 07, 2012 |
| Title |
MOV10 PAR-CLIP |
| Sample type |
SRA |
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| Source name |
MOV10 PAR-CLIP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
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| Treatment protocol |
HEK293 cells were incubated with 4-thiouridine over night
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| Growth protocol |
HEK293 cells were cultured in DMEM medium with 10% FBS at 37°C
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| Extracted molecule |
total RNA |
| Extraction protocol |
Using the Invitrogen Flp-In T-REx system, HA-Streptavidin tagged MOV10 was expressed in HEK 293 cells. The PARCLIP protocol by Hafner et al, Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP, 2010, Cell, was modified to allow for a nuclear isolation step prior to the immunoprecipitation as well as the use of the Streptavidin tag. After 365 nm crosslinking the cells were harvested, washed with cold PBS and pelleted at 500 rcf for 5 minutes. The supernatant was removed and every 6x106 cells were resuspended in a cold mixture of 3 mL of PBS, 9 mL of mQ water and 3 mL of Nuclear Isolation Buffer (1.28 M Sucrose, 40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 4 % Triton X-100) with Aprotinin 3.3 μg/mL, Leupeptin 10 μg/mL, Pepstatin 4 μg/mL added. The resulting suspension was rolled for 10 minutes at 4°C and subsequently spun at 2500 rcf for 10 minutes. After aspirating the supernatant, nuclei were resuspended in 1 mL of Lysis Buffer (50 mM HEPES pH 7.5, 150 mM KCl, 1 mM NaF, 1 % NP40, Roche cOmplete Proteinase Inhibitors) for every 6x106 cells. The resuspended nuclei were sonicated in a diagenode Bioruptor for 15 cycles of 30 seconds on and 30 seconds off. Except for minor changes, all subsequent steps followed the protocol in the original protocoll (Hafner et al. see above) library as obtained by the method described above
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
quality filtering according to the Illumina quality filter
adapter removal. The adapter introduced during the procedure required for sequencing is removed
Bowtie alignment using the following parameter setting:--best --chunkmbs 512 -S -M 100 -p 8 -k 1
Aligned reads were used to compute the relative substitution frequency at positions with coverage of at least 20
Genome_build: hg19
Supplementary_files_format_and_content: The final file contains information about read substitutions with respect to the reference genome at specific genomic positions. Each row specificies one substitution. The columns contain information regarding chromosome, genomic position (zero offset), strand, substitution, coverage at position and number of specified substitions at the position
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| Submission date |
Apr 23, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Cem Sievers |
| E-mail(s) |
cem.sievers@bsse.ethz.ch
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| Organization name |
ETH Zurich
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| Street address |
Mattenstrasse
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| City |
Basel |
| ZIP/Postal code |
4057 |
| Country |
Switzerland |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE37524 |
Mixture models and wavelet transforms reveal high confidence RNA-protein interaction sites in MOV10 PAR-CLIP data |
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| Relations |
| SRA |
SRX144163 |
| BioSample |
SAMN00862540 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM921128_mov10_cov20_completeData.txt.gz |
989.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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