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| Status |
Public on Sep 11, 2014 |
| Title |
U2af65-PAR-iCLIP_nSR100-KD |
| Sample type |
SRA |
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| Source name |
N2A, nSR100 depleted
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| Organism |
Mus musculus |
| Characteristics |
cell line: N2A
shRNA/sirna: shRNA against 3'UTR of nSR100
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| Treatment protocol |
Cells were transduced with lentiviruses expressing shRNAs targeting nSR100. Stable populations were selected using 2 ug/mL puromycin. For PAR-iCLIP experiments, cells were treated with 100 uM 4-thiouridine, and crosslinked (0.15 J/cm2) at 365 nm in a Stratalinker 1800
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| Growth protocol |
N2A and 293T cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), sodium pyruvate, and non-essential amino acids. N2A and 293T cells that were transduced with lentiviruses were additionally cultured in the presence of 2ug/mL puromyocin to maintain selection.
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| Extracted molecule |
total RNA |
| Extraction protocol |
PAR-iCLIP was performed as described in Huppertz et. al, 2014, Methods.
PAR-iCLIP libraries were generated as described in Huppertz et. al, 2014, Methods
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
PAR-iCLIP barcode: NNTGGCNNN. Antibody: anti-U2af65
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| Data processing |
Full genomic sequences for the analyzed species were downloaded from the UCSC Genome Browser database. Full transcriptomic sequences for all species were downloaded from Ensembl. For each gene, the canonical transcript was selected for gene expression analysis based on BioMart.
For each species, we assembled all canonical transcript (as defined above) sequences. In order to calculate the effective number of unique mappable positions in each transcript (i.e. the effective length) we performed the following steps. For each read length k, we extracted the L-k+1 (L being the transcript length) k-mer sequences from each canonical transcript and then aligned the full set of k-mers against the respective genome using Bowtie, allowing for a maximum of two mismatches. k-mers with no or one unique genomic alignment were then likewise aligned back to the canonical transcriptome. For each transcript, the number of such k-mers having a unique transcriptomic alignment was determined. This corresponds to the transcript’s effective number of unique mappable positions for k-mer mRNA-Seq reads. For each sample, the corresponding mRNA-Seq data were aligned against the respective genome using Bowtie, allowing for a maximum of two mismatches. Reads with one unique genomic alignment were then aligned against the canonical transcriptome and, for each transcript, the number of reads with one unique transcriptomic alignment were counted. Gene expression levels were determined as reads per thousand mappable positions of target transcript sequence per million of reads, where the reads uniquely align to the analyzed transcriptome. This procedure for estimating gene expression levels is a corrected version of the widely used RPKM (reads per kilobase of target transcript sequence per million of total reads) metric, and is referred to as “cRPKM” [Labbe RM et al. Stem Cells (2012)].
PAR-iCLIP: Reads were collapsed, and the barcode and adaptor sequences were trimmed from the 5' and 3' ends respectively. Tags longer than 20 nucleotides were mapped to the human reference geneome hg19 using Bowtie by allowing a maximum of 2 mismatches, and only tags with less than or equal to five mapped locations were kept. Bedgraphs were generated using the BEDtools (v2.17.0) tool: genomecov
Genome_build: mm9, hg19
Supplementary_files_format_and_content: Tab-delimited text files include cRPKM values (as defined above), as well as the total read count for each gene's canonical transcript. Bedgraphs of individual (PTBP1, U2af65) and pooled (nSR100, 3 biological replicates) PAR-iCLIP tag densities are provided
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| Submission date |
May 05, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Bushra Raj |
| E-mail(s) |
bushra.raj@pennmedicine.upenn.edu
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| Organization name |
University of Pennsylvania
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| Street address |
421 Curie Boulevard
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE57278 |
A global regulatory mechanism for activating an exon network required for neurogenesis |
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| Relations |
| BioSample |
SAMN02743683 |
| SRA |
SRX532489 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1378379_N2A_L4_U2af65v2.bedgraph.gz |
5.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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