it is known that phosphorylated srsf10 functions as a sequence - specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo , . to understand the global profile of the alternative splicing network regulated by srsf10 in vivo , we carried out an rna - seq approach to analyze the effects of srsf10 depletion on splicing regulation in chicken dt40 cells . the general strategy for sample preparation for rna - seq was provided in fig . 1a and the detailed experimental procedures were as follows . wild type ( wt ) and srsf10-knockout ( ko ) dt40 cells were cultured in a standard medium to 90% confluence . total rna was isolated from these cells , and cdna libraries were prepared individually by performing a series of experiments , including poly(a ) enrichment , rna fragmentation , random hexamer - primed cdna synthesis , linker ligation , size selection and pcr amplification . after selecting approximately 200-bp fragments by agarose gel electrophoresis , illumina paired - end sequencing adapters were ligated to the dna fragments for pcr amplification and sequencing with an illumina hiseq 2000 system . briefly , images generated by sequencers were converted by base calling into nucleotide sequences , which were called raw reads . then raw reads were trimmed for adaptor sequence , and masked for low - complexity or low - quality sequence to get clean reads with high quality . the clean reads were then aligned to the reference chicken genome ( release : galgal4 ) and the corresponding recently annotated genes ( galgal4.0 ) using the tophat program ( v2.0.6 ) . in the alignment , preliminary experiments were performed to optimize the alignment parameters to provide the most information on the alternative splicing events . cufflinks   was used to construct ko and wt dt40 transcriptomes and cuffmerge merges the transcriptomes with the reference gene annotations ( galgal4.0 ) to generate a new comprehensive transcriptome . finally , a java program called asd ( alternative splicing detector , available at http://www.novelbio.com/asd/asd.html ) was developed to detect and assess alternative splicing events between the two samples . there are five major modes of alternative splicing ( as ) events described in metazoan organisms , including cassette exon , alternative 3 splice site , alternative 5 splice site , mutually exclusive exon and retained intron ( fig . , we were able to identify 167 events belonging to the five major modes of as from a total number of 10,652 splicing events that changed significantly between the wt and the ko samples by asd . specifically , the majority of the affected splicing events ( 130 ) belonged to the cassette exon category . strikingly , analytical results by asd indicated that srsf10 could both activate and repress exon inclusion ( fig . in addition , the selection of alternative 3 and 5 splice sites was similarly affected by srsf10 . taken together , these results provide strong evidence that srsf10 can promote both exon inclusion and exclusion in vivo . to understand how loss of srsf10 affects cellular biological processes , we next performed gene ontology ( go ) and pathway analysis of srsf10-affected genes by using cluego . function enrichment analysis indicated that these affected spliced genes are involved in multiple processes , including histone methylation , response to external stimulus , glucose metabolic processes and mitochondrion distribution ( fig . these results illustrated that srsf10 might play an important role in a wide range of biological processes by controlling its downstream splicing targets . it is known that phosphorylated srsf10 functions as a sequence - specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo , . to understand the global profile of the alternative splicing network regulated by srsf10 in vivo , we carried out an rna - seq approach to analyze the effects of srsf10 depletion on splicing regulation in chicken dt40 cells . the general strategy for sample preparation for rna - seq was provided in fig . 1a and the detailed experimental procedures were as follows . wild type ( wt ) and srsf10-knockout ( ko ) dt40 cells were cultured in a standard medium to 90% confluence . total rna was isolated from these cells , and cdna libraries were prepared individually by performing a series of experiments , including poly(a ) enrichment , rna fragmentation , random hexamer - primed cdna synthesis , linker ligation , size selection and pcr amplification . after selecting approximately 200-bp fragments by agarose gel electrophoresis , illumina paired - end sequencing adapters were ligated to the dna fragments for pcr amplification and sequencing with an illumina hiseq 2000 system . briefly , images generated by sequencers were converted by base calling into nucleotide sequences , which were called raw reads . then raw reads were trimmed for adaptor sequence , and masked for low - complexity or low - quality sequence to get clean reads with high quality . the clean reads were then aligned to the reference chicken genome ( release : galgal4 ) and the corresponding recently annotated genes ( galgal4.0 ) using the tophat program ( v2.0.6 ) . in the alignment , preliminary experiments were performed to optimize the alignment parameters to provide the most information on the alternative splicing events . cufflinks   was used to construct ko and wt dt40 transcriptomes and cuffmerge merges the transcriptomes with the reference gene annotations ( galgal4.0 ) to generate a new comprehensive transcriptome . finally , a java program called asd ( alternative splicing detector , available at http://www.novelbio.com/asd/asd.html ) was developed to detect and assess alternative splicing events between the two samples . there are five major modes of alternative splicing ( as ) events described in metazoan organisms , including cassette exon , alternative 3 splice site , alternative 5 splice site , mutually exclusive exon and retained intron ( fig . , we were able to identify 167 events belonging to the five major modes of as from a total number of 10,652 splicing events that changed significantly between the wt and the ko samples by asd . specifically , the majority of the affected splicing events ( 130 ) belonged to the cassette exon category . strikingly , analytical results by asd indicated that srsf10 could both activate and repress exon inclusion ( fig . in addition , the selection of alternative 3 and 5 splice sites was similarly affected by srsf10 . taken together , these results provide strong evidence that srsf10 can promote both exon inclusion and exclusion in vivo . to understand how loss of srsf10 affects cellular biological processes , we next performed gene ontology ( go ) and pathway analysis of srsf10-affected genes by using cluego . function enrichment analysis indicated that these affected spliced genes are involved in multiple processes , including histone methylation , response to external stimulus , glucose metabolic processes and mitochondrion distribution ( fig .  3 ) . these results illustrated that srsf10 might play an important role in a wide range of biological processes by controlling its downstream splicing targets . we present here a unique dataset of rna - seq results in which we globally analyzed srsf10-regulated as events based on deep sequencing of the chicken transcriptome . we found that srsf10 is involved in all of the common modes of as , with cassette exons being the most frequent targets for srsf10 regulation . finally , srsf10-affected splicing genes were involved in multiple processes , indicating a role of srsf10 in these processes . this dataset is of high quality and will facilitate future study of srsf10 function in vivo and will also improve understanding of as regulation in vivo .
splicing factor srsf10 is known to function as a sequence - specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo . 
 we recently used an rna - seq approach coupled with bioinformatics analysis to identify the extensive splicing network regulated by srsf10 in chicken cells . 
 we found that srsf10 promoted both exon inclusion and exclusion . 
 functionally , many of the srsf10-verified alternative exons are linked to pathways of response to external stimulus . here 
 we describe in detail the experimental design , bioinformatics analysis and go / pathway enrichment analysis of srsf10-regulated genes to correspond with our data in the gene expression omnibus with accession number gse53354 . 
 our data thus provide a resource for studying regulation of alternative splicing in vivo that underlines biological functions of splicing regulatory proteins in cells .