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Sample GSM1378376

Query DataSets for GSM1378376

Status

Public on Sep 11, 2014

Title

nSR100 PAR-iCLIP rep3

Sample type

SRA

Source name

293T, dox-inducible

Organism

Homo sapiens

Characteristics

cell line: 293T

Treatment protocol

A stable cell line was established by transducing cells with dox-inducible lentiviruses expressing Flag-nSR100 and selection with 2 ug/mL puromycin. For PAR-iCLIP experiments, cells were treated with 2 ug/mL dox, 100 uM 4-thiouridine, and crosslinked (0.15 J/cm2) at 365 nm in a Stratalinker 1800

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.

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

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

PAR-iCLIP barcode: NNTTAANNN. Antibody: anti-FLAG
293TnSR100_PariCLIP_pool2.bedgraph

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

Submission date

May 05, 2014

Last update date

May 15, 2019

Contact name

Bushra Raj

E-mail(s)

bushraraj@fas.harvard.edu

Organization name

Harvard University

Street address

16 Divinity Avenue