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Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data.

Misra A, Ou J, Zhu LJ, Green MR - Genom Data (2015)

Bottom Line: Alternative splicing is a key mechanism for generating proteome diversity, however the mechanisms regulating alternative splicing are poorly understood.Using a genome-wide RNA interference screening strategy, we identified cleavage and polyadenylation specificity factor (CPSF) and symplekin (SYMPK) as cofactors of the well-known splicing regulator RBFOX2.Here, we describe the experimental design, and the quality control and data analyses that were performed on the dataset.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, USA ; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

ABSTRACT
Alternative splicing is a key mechanism for generating proteome diversity, however the mechanisms regulating alternative splicing are poorly understood. Using a genome-wide RNA interference screening strategy, we identified cleavage and polyadenylation specificity factor (CPSF) and symplekin (SYMPK) as cofactors of the well-known splicing regulator RBFOX2. To determine the role of CPSF in alternative splicing on a genome-wide level, we performed paired-end RNA sequencing (RNA-seq) to compare splicing events in control cells and RBFOX2 or CPSF2 knockdown cells. We also performed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify direct binding targets of RBFOX2 and CPSF2. Here, we describe the experimental design, and the quality control and data analyses that were performed on the dataset. The raw sequencing data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE60392.

No MeSH data available.


Related in: MedlinePlus

Graphs representing per base quality using Phred score for the iCLIP data after cleanup. The figure is generated by fastQC.
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f0005: Graphs representing per base quality using Phred score for the iCLIP data after cleanup. The figure is generated by fastQC.

Mentions: Two biological replicates of the RBFOX2 and CPSF2 iCLIP libraries were sequenced with a single-end length of 100 bp. Only reads containing the 5′ adapter were considered for analysis. The 3′ and 5′ adaptor sequences were trimmed from these reads. Furthermore, ribosomal RNA reads were filtered out using SortMeRNA(v1.9). The RNA database used by SortMeRNA was downloaded from http://www.arb-silva.de/ [2]. Homopolymer sequences > 10 bp were removed followed by sequence quality assessment using fastQC (v 0.10.1) [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/] (Fig. 1). After clean up, the read lengths were in the range of 21–72 bp. The fastQC score for all the bases was > 30, indicating that the reads were of high quality. Quality assessment was also performed on raw reads before clean up and similar quality was observed. Following cleanup, the reads were mapped against human reference genome (GRCh37/hg19, Feb. 2009) using bowtie (v1.0.0) [3] with the following parameter setting: “-q -e 100 -l 20 -m 1 –best –strata”. Finally, the iCLIP-seq reads were collapsed to remove PCR artifacts using picard tools (v1.94). Reproducibility of the biological replicates was assessed using Pearson correlation analysis (RBFOX2 r = 0.825, p-value < 2.2e − 16; CPSF2 r = 0.824, p-value < 2.2e − 16) (see Fig. S2D in [4]).


Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data.

Misra A, Ou J, Zhu LJ, Green MR - Genom Data (2015)

Graphs representing per base quality using Phred score for the iCLIP data after cleanup. The figure is generated by fastQC.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664754&req=5

f0005: Graphs representing per base quality using Phred score for the iCLIP data after cleanup. The figure is generated by fastQC.
Mentions: Two biological replicates of the RBFOX2 and CPSF2 iCLIP libraries were sequenced with a single-end length of 100 bp. Only reads containing the 5′ adapter were considered for analysis. The 3′ and 5′ adaptor sequences were trimmed from these reads. Furthermore, ribosomal RNA reads were filtered out using SortMeRNA(v1.9). The RNA database used by SortMeRNA was downloaded from http://www.arb-silva.de/ [2]. Homopolymer sequences > 10 bp were removed followed by sequence quality assessment using fastQC (v 0.10.1) [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/] (Fig. 1). After clean up, the read lengths were in the range of 21–72 bp. The fastQC score for all the bases was > 30, indicating that the reads were of high quality. Quality assessment was also performed on raw reads before clean up and similar quality was observed. Following cleanup, the reads were mapped against human reference genome (GRCh37/hg19, Feb. 2009) using bowtie (v1.0.0) [3] with the following parameter setting: “-q -e 100 -l 20 -m 1 –best –strata”. Finally, the iCLIP-seq reads were collapsed to remove PCR artifacts using picard tools (v1.94). Reproducibility of the biological replicates was assessed using Pearson correlation analysis (RBFOX2 r = 0.825, p-value < 2.2e − 16; CPSF2 r = 0.824, p-value < 2.2e − 16) (see Fig. S2D in [4]).

Bottom Line: Alternative splicing is a key mechanism for generating proteome diversity, however the mechanisms regulating alternative splicing are poorly understood.Using a genome-wide RNA interference screening strategy, we identified cleavage and polyadenylation specificity factor (CPSF) and symplekin (SYMPK) as cofactors of the well-known splicing regulator RBFOX2.Here, we describe the experimental design, and the quality control and data analyses that were performed on the dataset.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, USA ; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

ABSTRACT
Alternative splicing is a key mechanism for generating proteome diversity, however the mechanisms regulating alternative splicing are poorly understood. Using a genome-wide RNA interference screening strategy, we identified cleavage and polyadenylation specificity factor (CPSF) and symplekin (SYMPK) as cofactors of the well-known splicing regulator RBFOX2. To determine the role of CPSF in alternative splicing on a genome-wide level, we performed paired-end RNA sequencing (RNA-seq) to compare splicing events in control cells and RBFOX2 or CPSF2 knockdown cells. We also performed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify direct binding targets of RBFOX2 and CPSF2. Here, we describe the experimental design, and the quality control and data analyses that were performed on the dataset. The raw sequencing data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE60392.

No MeSH data available.


Related in: MedlinePlus