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| Status |
Public on Jul 15, 2013 |
| Title |
CLIP_noemetine_SigmaAb |
| Sample type |
SRA |
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| Source name |
293S cells
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| Organism |
Homo sapiens |
| Characteristics |
treatment: no treatment (control)
cell line: 293S
antibody: Ago2
antibody manufacturer: Sigma
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| Treatment protocol |
Where indicated, treated with 20 μg/mL emetine for 60 minutes
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| Growth protocol |
293S cells were grown in DMEM + 10% FBS + penicillin/streptomycin, in adherent monolayer, to ~70% confluency.
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| Extracted molecule |
total RNA |
| Extraction protocol |
CLIP-seq - Ago2 crosslinking-immunoprecipitation of RNA
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Ago2-crosslinked RNA
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| Data processing |
Basecalling with Illumina RTA 1.8.70.0
Sequenced reads were trimmed of the fixed 3` adaptor and 5` adaptor sequences. The 5` RNA adaptor contains a 4-nucleotide random region (a "counting" barcode) that serves to count distinct 5` ligation events in the library preparation. The count of such unique barcodes associated with each unique CLIPped RNA fragment (the insert sequence without adaptors) was used as its readcount, instead of the total read multiplicity. This avoids PCR amplification bias in the quantitation. The insert sequences without adaptors were mapped to the hg19 human genome assembly using Bowtie version 0.12.8 iteratively with 0, 1, and 2 mismatches, retaining the mapped reads from each stage and passing the non-mappers to the next stage, only allowing unique mappers. Reads that did not map to the genome were processed with Tophat to identify reads mapping to RefSeq RNA and lincRNA splice junctions. Reads mapping to identical genomic positions were collapsed and summed. Combined reads were annotated for RefSeq, lincRNA, microRNA and RepeatMasker categories. To identify peaks, reads from all replicates for each condition were pooled and used as input to Findpeaks version 4.0. Parameters were set to denote peaks separated by valleys of 50% height or less, and to trim the sides of peaks dropping below 10% of peak height. To identify peaks from two conditions (eg., +/- arsenite) that correspond to the same Ago2 binding site, while allowing for some differences (shifting) in their end coordinates, peaks from the two conditions that overlapped more than 50% of their width were designated as the same peak/binding location, and the union of their coordinates was used as the genomic coordinates for this "peak pair" or "combined peak". This included pseudo-pairs, with a peak present in one condition and completely absent in the other. Annotations for peaks were obtained as above, and the coordinates of combined peaks were used to re-extract readcounts from individual replicates under the peak. Readcounts for the corresponding mRNAs from RNA-seq replicates were appended to the dataset, and peaks were filtered for the presence of greater than 10 readcounts across all CLIP replicates in an experiment (treatment and control), as well as greater than 10 readcounts of the corresponding mRNA. CLIP readcounts were then normalized to the miRNA fraction (separately for each replicate), and RNA-seq readcounts were normalized across replicates by applying DESeq-derived sizing factors. Normalized replicate counts were padded with a value of 0.25, and the CLIP signal for each peak was then divided by the corresponding mRNA abundance in the replicate.
hg19
tab-delimited text file with Ago2 CLIP peak genomic coordinates, annotation and normalized natural log abundance and logratio of change in treatment
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| Submission date |
Feb 18, 2013 |
| Last update date |
Mar 04, 2023 |
| Contact name |
Fedor Karginov |
| E-mail(s) |
karginov@ucr.edu
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| Organization name |
UC Riverside
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| Department |
MCSB
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| Street address |
900 University Ave
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| City |
Riverside |
| State/province |
California |
| ZIP/Postal code |
92521 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (2) |
| GSE44377 |
Remodeling of Ago2-mRNA interactions upon cellular stress reflects miRNA complementarity and correlates with altered translation rates (part 2) |
| GSE44404 |
Remodeling of Ago2-mRNA interactions upon cellular stress reflects miRNA complementarity and correlates with altered translation rates |
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| Relations |
| SRA |
SRX241586 |
| BioSample |
SAMN01922204 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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