the iclip and rna - seq data are available at the following geo series : gse60392 , http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60392.celltreatmentfeaturereplicatesgeo accession urlhuman flp - in 293untreatedrbfox2 iclip2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477476human flp - in 293untreatedcpsf2 iclip2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477475human flp - in 293non - silencing lentivirus knockdownpaired - end rna - seq2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477603http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477604human flp - in 293rbfox2 lentivirus knockdownpaired - end rna - seq2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477601http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477602human flp - in 293cpsf2 lentivirus knockdownpaired - end rna - seq2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477599http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477600
cells were transduced with lentiviruses expressing shrnas targeting rbfox2 or cpsf2 , or as a control a non - silencing ( ns ) shrna , and then puromycin selected for 4  days .
flp - in-293 cells were grown in 10  cm plates and then subjected to uv - c irradiation ( 200  mj / cm , stratalinker 2400 ) . upon removal of phosphate buffered saline ( pbs ) , cells were scraped off and precipitated by centrifugation at 3000  rpm for 10  min .
hcl ph  7.4 , 100  mm nacl , 1  mm mgcl2 , 0.1  mm cacl2 , 1% np-40 , 0.1% sds , 0.5% sodium deoxycholate ) .
samples were partially digested with rnase i by incubation with 10  l rnase i ( ambion ) ( diluted 1:500 in lysis buffer ) and 5  l turbo dnase ( ambion ) for 5  min at 37  c while shaking at 1100  rpm .
cells were centrifuged at 14,000  rpm for 15  min to collect the cross - linked lysate . to prepare
the beads for rna purification , 50  l of protein g - coated dynabeads ( invitrogen ) was washed twice with 1  ml lysis buffer and resuspended in 200  l lysis buffer containing 35  g of rbfox2 ( bethyl laboratories , a300 - 864a ) or cpsf2 ( bethyl laboratories , a301 - 580a ) antibody .
the beads were rotated at room temperature for 60  min , washed twice with lysis buffer , and added to the cross - linked lysate .
after overnight incubation at 4  c , the beads were washed four times with high - salt wash buffer ( 50  mm tris  hcl ph  7.4 , 1  m nacl , 1  mm edta , 0.1% sds , 0.5% sodium deoxycholate , 1% np-40 ) and once with pnk wash buffer ( 20  mm tris  hcl ph  7.4 , 10  mm mgcl2 , 0.2% tween-20 ) .
the beads were resuspended in 5  l 10   pnk buffer , 44  l h2o and 1  l pnk enzyme ( neb ) and incubated at 37  c for 20  min while shaking at 1100  rpm .
samples were then washed twice with high - salt wash buffer and once with 900  l pnk wash buffer .
to ligate linkers to the 3 end of rnas , 4.5  l of purified rna was mixed with 1  l of 10  m mircat33 adaptor ( integrated dna technologies ) , 1.5  l of rna ligase buffer , 0.5  l of t4 rna ligase 2 , truncated k227q ( neb ) , and 7.5  l of peg 8000 , and incubated at 30  c for 6  h. samples were then end - labeled using pnk enzyme and run on a nupage novex 10% bis  tris protein gel ( invitrogen ) with 1   mops running buffer ( invitrogen ) .
proteins and covalently bound rnas were then transferred to a nitrocellulose membrane ( whatman ) using a novex wet transfer apparatus ( invitrogen ) .
the nitrocellulose membrane was rinsed with 1   pbs , wrapped in plastic wrap and exposed to x - ray film . to isolate the rna 
protein complex , the region of the nitrocellulose membrane corresponding to the complex size of interest was cut out .
cross - linked rnas were then isolated by incubating the nitrocellulose membrane pieces with pk buffer ( 100  mm tris  hcl ph  7.5 , 50  mm nacl , 10  mm edta ) containing 2  mg / ml proteinase k ( roche ) for 20  min at 37  c , after which an equal volume of pk buffer containing 7  m urea was added to the tube and the samples were incubated for another 20  min at 37  c .
subsequently , 600  l of phenol / chloroform ( ambion ) was added , and the mixture was incubated for 10  min at room temperature .
samples were then centrifuged for 10  min at 13,000  rpm at room temperature , and the aqueous phase was transferred into a new microtube and again centrifuged .
400  l of supernatant was mixed with 0.5  l glycoblue ( ambion ) , 40  l 3  m sodium acetate ph  5.5 , and 1  ml 100% ethanol and incubated overnight at   20  c .
rnas were precipitated by centrifugation for 30  min at 15,000  rpm at 4  c , washed with 500  l 80% ethanol and resuspended in 30  l h2o . to carry out reverse transcription ,
30  l purified rna was mixed with 1  l of either bc2 ( 5-pggcgatgagatcggaagagcgtcgtgtagggaaagagtgt - sp18-ctcg gcattcctgctgaaccgctcttccgatct - ccttggcacccgagaattcca-3 ) or bc3 ( 5-pggcgatgagatcggaagagcgtcgtgtagggaaagagtgt - sp18-ctcg gcattcctgctgaaccgctcttccgatct - ccttggcacccgagaattcca-3 ) prt primer ( 10  m concentration ) and 1.5  l 10  mm dntp mix , and incubated at 65  c for 5  min and then transferred to ice . subsequently ,
9  l of 5   first strand synthesis buffer ( invitrogen ) , 2.25  l 0.1  m dtt , and 1.25  l superscript iii reverse transcriptase ( invitrogen ) were added to a final volume of 45  l .
the cdnas were size - separated on a 6% tbe urea gel ( invitrogen ) .
two size fractions were recovered from the gel by cutting the gel corresponding to a cdna size of 120155  nt and 150225  nt .
gel fragments were mixed with 400  l te buffer , crushed with a 1  ml syringe plunger and centrifuged overnight at 1100  rpm at 37  c .
gel pieces and the supernatant were separated using a costar spinx column ( corning incorporated ) . 40  l 3  m sodium acetate ph  5.5 and 0.5  l glycogen were added to the supernatant and centrifuged at 13,000  rpm at   20  c overnight to precipitate the reverse transcription product .
the cdna samples were then circularized by mixing with 2.0  l 10   circligase buffer ( epicentre ) , 1  l 50  mm mncl2 and 1  l circligase ii ( epicentre ) and incubated for 34  h at 60  c . for high - throughput sequencing ,
briefly , cdnas were mixed with 1  m each of pe1.0 ( 5-aatgatacggcgaccaccg agatctacactctttccctacacgacgctcttccgatct-3 ) and pe2.0 ( 5-caagc agaagacggcatacgagatcggtctcggcattcctgctgaaccgctcttccgatct ) primers ( 10  m concentration ) and kapa 2   ready mix ( kapa biosystems ) , and pcr amplification was performed as per the manufacturer 's instructions .
the pcr product was gel eluted and subjected to sequencing on a hiseq 2000 sequencing system ( illumina ) .
two biological replicates of the rbfox2 and cpsf2 iclip libraries were sequenced with a single - end length of 100  bp
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 2172  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 ,
2009 ) using bowtie ( v1.0.0 )   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 .
rna - seq samples were prepared using a truseq rna library prep kit v2 ( illumina ) according to the manufacturer 's instructions .
sequencing was done using an illumina hiseq 2000 with a paired - end length of 100  bp for duplicated ns , rbfox2- knockdown and cpsf2-knockdown samples .
the fastqc score for all the bases was >  30 , indicating that the reads were of high quality .
2009 ) using tophat ( v2.0.9 , bowtie2/2.1.0 )   with the following parameter setting :  -g [ ucsc_hg19_knowngene ] mate - inner - dist 50 b2-very - sensitive  .
the ucsc_hg19_knowngene annotation file was downloaded from ucsc table browser and quality of alignment was assessed using samstat ( v 1.09 ) .
a pie chart describing the quality of sequence alignment distribution showed that >  97% of the reads have mapq    30 , indicating the high mapping quality of the reads ( fig .
gene expression level ( fpkm ) and differential gene expression analysis were performed using cufflinks ( v2.1.1 ) .
python script , provided by the dexseq ( v1.10.6 ) package , was used for exon level read count estimation with the following parameter setting : 
pearson correlation analysis of gene expression levels was performed to evaluate the reproducibility between biological replicates ( ns : r  =  0.991 , p - value  <  2.2e    16 ; rbfox2 : r  =  0.991 , p - value  <  2.2e    16 ; cpsf2 : r  =  0.992 , p - value  <  2.2e    16 ) ( fig .
s2a in ) pearson correlation analysis of exon level expression also demonstrated high reproducibility between biological replicates ( ns : r  =  0.997 , p - value  <  2.2e    16 ; rbfox2 : r  =  0.999 , p - value  <  2.2e    16 ; cpsf2 : r  =  0.989 , p - value  <  2.2e    16 ) ( fig .
multidimensional scaling ( mds ) plot was generated to visualize the similarity of gene expression between biological replicates and dissimilarity among ns , rbfox2 and cpsf2 knockdown samples using cummerbund package ( v 2.8.2 ) .
the results of the mds plot showed that the biological replicates clustered closely while there was a clear segregation among ns , rbfox2 and cpsf2 knockdown samples , indicating that biological replicates were similar to each other and different knockdown groups had different expression profiles ( fig . 2c ) . to visualize the distribution of gene expression level for each sample , a boxplot was generated for each of the samples using log10 transformed fpkm values from cufflinks ( fig .
the quartiles and overall range were consistent between biological replicates , indicating that the data were reproducible and of high quality .
alternative splicing events were identified using the dexseq ( v1.10.6 ) package , which models the exon counts as the negative binomial distribution and uses generalized linear models to test differential exon - usage using variance estimated from biological replicates .
sequence depths were used to normalize the exon counts for each sample followed by dispersion estimation before detecting alternative splicing events .
dispersion plots showed that most of the final exon level dispersions follow the fitted line as expected ( fig .
exons with a false discovery rate  <  0.05 , log2 fold change greater than 1.2 , and a minimum of 10 counts for at least one of the samples were considered differentially expressed .
alternative splicing events with chromosome coordinates overlapping with constitutive exons ( ensemble release version 75 ) by at least 1  bp were filtered out using the bioconductor genomicranges ( v 1.18.1 ) package .
exons shorter than 12  bp or belonging to differentially expressed genes were filtered out from the list .
dexseq creates a set of disjoint exon fragments ( pseudo exon ) from the ensemble exon annotation file .
cells were transduced with lentiviruses expressing shrnas targeting rbfox2 or cpsf2 , or as a control a non - silencing ( ns ) shrna , and then puromycin selected for 4  days .
flp - in-293 cells were grown in 10  cm plates and then subjected to uv - c irradiation ( 200  mj / cm , stratalinker 2400 ) . upon removal of phosphate buffered saline ( pbs ) , cells were scraped off and precipitated by centrifugation at 3000  rpm for 10  min .
hcl ph  7.4 , 100  mm nacl , 1  mm mgcl2 , 0.1  mm cacl2 , 1% np-40 , 0.1% sds , 0.5% sodium deoxycholate ) .
samples were partially digested with rnase i by incubation with 10  l rnase i ( ambion ) ( diluted 1:500 in lysis buffer ) and 5  l turbo dnase ( ambion ) for 5  min at 37  c while shaking at 1100  rpm .
cells were centrifuged at 14,000  rpm for 15  min to collect the cross - linked lysate . to prepare
the beads for rna purification , 50  l of protein g - coated dynabeads ( invitrogen ) was washed twice with 1  ml lysis buffer and resuspended in 200  l lysis buffer containing 35  g of rbfox2 ( bethyl laboratories , a300 - 864a ) or cpsf2 ( bethyl laboratories , a301 - 580a ) antibody .
the beads were rotated at room temperature for 60  min , washed twice with lysis buffer , and added to the cross - linked lysate .
after overnight incubation at 4  c , the beads were washed four times with high - salt wash buffer ( 50  mm tris 
hcl ph  7.4 , 1  m nacl , 1  mm edta , 0.1% sds , 0.5% sodium deoxycholate , 1% np-40 ) and once with pnk wash buffer ( 20  mm tris  hcl ph  7.4 , 10  mm mgcl2 , 0.2% tween-20 ) .
the beads were resuspended in 5  l 10   pnk buffer , 44  l h2o and 1  l pnk enzyme ( neb ) and incubated at 37  c for 20  min while shaking at 1100  rpm .
samples were then washed twice with high - salt wash buffer and once with 900  l pnk wash buffer . to ligate linkers to the 3 end of rnas
, 4.5  l of purified rna was mixed with 1  l of 10  m mircat33 adaptor ( integrated dna technologies ) , 1.5  l of rna ligase buffer , 0.5  l of t4 rna ligase 2 , truncated k227q ( neb ) , and 7.5  l of peg 8000 , and incubated at 30  c for 6  h. samples were then end - labeled using pnk enzyme and run on a nupage novex 10% bis  tris protein gel ( invitrogen ) with 1   mops running buffer ( invitrogen ) .
proteins and covalently bound rnas were then transferred to a nitrocellulose membrane ( whatman ) using a novex wet transfer apparatus ( invitrogen ) .
the nitrocellulose membrane was rinsed with 1   pbs , wrapped in plastic wrap and exposed to x - ray film . to isolate the rna 
protein complex , the region of the nitrocellulose membrane corresponding to the complex size of interest was cut out .
cross - linked rnas were then isolated by incubating the nitrocellulose membrane pieces with pk buffer ( 100  mm tris  hcl ph  7.5 , 50  mm nacl , 10  mm edta ) containing 2  mg / ml proteinase k ( roche ) for 20  min at 37  c , after which an equal volume of pk buffer containing 7  m urea was added to the tube and the samples were incubated for another 20  min at 37  c .
subsequently , 600  l of phenol / chloroform ( ambion ) was added , and the mixture was incubated for 10  min at room temperature .
samples were then centrifuged for 10  min at 13,000  rpm at room temperature , and the aqueous phase was transferred into a new microtube and again centrifuged .
400  l of supernatant was mixed with 0.5  l glycoblue ( ambion ) , 40  l 3  m sodium acetate ph  5.5 , and 1  ml 100% ethanol and incubated overnight at   20  c .
rnas were precipitated by centrifugation for 30  min at 15,000  rpm at 4  c , washed with 500  l 80% ethanol and resuspended in 30  l h2o . to carry out reverse transcription ,
30  l purified rna was mixed with 1  l of either bc2 ( 5-pggcgatgagatcggaagagcgtcgtgtagggaaagagtgt - sp18-ctcg gcattcctgctgaaccgctcttccgatct - ccttggcacccgagaattcca-3 ) or bc3 ( 5-pggcgatgagatcggaagagcgtcgtgtagggaaagagtgt - sp18-ctcg gcattcctgctgaaccgctcttccgatct - ccttggcacccgagaattcca-3 ) prt primer ( 10  m concentration ) and 1.5  l 10  mm dntp mix , and incubated at 65  c for 5  min and then transferred to ice .
subsequently , 9  l of 5   first strand synthesis buffer ( invitrogen ) , 2.25  l 0.1  m dtt , and 1.25  l superscript iii reverse transcriptase ( invitrogen ) were added to a final volume of 45  l .
the cdnas were size - separated on a 6% tbe urea gel ( invitrogen ) .
two size fractions were recovered from the gel by cutting the gel corresponding to a cdna size of 120155  nt and 150225  nt .
gel fragments were mixed with 400  l te buffer , crushed with a 1  ml syringe plunger and centrifuged overnight at 1100  rpm at 37  c .
gel pieces and the supernatant were separated using a costar spinx column ( corning incorporated ) . 40  l 3  m sodium acetate ph  5.5 and 0.5  l glycogen were added to the supernatant and centrifuged at 13,000  rpm at   20  c overnight to precipitate the reverse transcription product .
the cdna samples were then circularized by mixing with 2.0  l 10   circligase buffer ( epicentre ) , 1  l 50  mm mncl2 and 1  l circligase ii ( epicentre ) and incubated for 34  h at 60  c . for high - throughput sequencing ,
briefly , cdnas were mixed with 1  m each of pe1.0 ( 5-aatgatacggcgaccaccg agatctacactctttccctacacgacgctcttccgatct-3 ) and pe2.0 ( 5-caagc agaagacggcatacgagatcggtctcggcattcctgctgaaccgctcttccgatct ) primers ( 10  m concentration ) and kapa 2   ready mix ( kapa biosystems ) , and pcr amplification was performed as per the manufacturer 's instructions .
the pcr product was gel eluted and subjected to sequencing on a hiseq 2000 sequencing system ( illumina ) .
two biological replicates of the rbfox2 and cpsf2 iclip libraries were sequenced with a single - end length of 100  bp
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 .
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 ,
2009 ) using bowtie ( v1.0.0 )   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 .
rna - seq samples were prepared using a truseq rna library prep kit v2 ( illumina ) according to the manufacturer 's instructions .
sequencing was done using an illumina hiseq 2000 with a paired - end length of 100  bp for duplicated ns , rbfox2- knockdown and cpsf2-knockdown samples .
the fastqc score for all the bases was >  30 , indicating that the reads were of high quality .
2009 ) using tophat ( v2.0.9 , bowtie2/2.1.0 )   with the following parameter setting :  -g [ ucsc_hg19_knowngene ] mate - inner - dist 50 b2-very - sensitive  .
the ucsc_hg19_knowngene annotation file was downloaded from ucsc table browser and quality of alignment was assessed using samstat ( v 1.09 ) .
a pie chart describing the quality of sequence alignment distribution showed that >  97% of the reads have mapq    30 , indicating the high mapping quality of the reads ( fig .
2a ) . gene expression level ( fpkm ) and differential gene expression analysis were performed using cufflinks ( v2.1.1 ) .
python script , provided by the dexseq ( v1.10.6 ) package , was used for exon level read count estimation with the following parameter setting : 
pearson correlation analysis of gene expression levels was performed to evaluate the reproducibility between biological replicates ( ns : r  =  0.991 , p - value  <  2.2e    16 ; rbfox2 : r  =  0.991 , p - value  <  2.2e    16 ; cpsf2 : r  =  0.992 , p - value  <  2.2e    16 ) ( fig . s2a in )
pearson correlation analysis of exon level expression also demonstrated high reproducibility between biological replicates ( ns : r  =  0.997 , p - value  <  2.2e    16 ; rbfox2 : r  =  0.999 , p - value  <  2.2e    16 ; cpsf2 : r  =  0.989 , p - value  <  2.2e    16 ) ( fig .
multidimensional scaling ( mds ) plot was generated to visualize the similarity of gene expression between biological replicates and dissimilarity among ns , rbfox2 and cpsf2 knockdown samples using cummerbund package ( v 2.8.2 ) .
the results of the mds plot showed that the biological replicates clustered closely while there was a clear segregation among ns , rbfox2 and cpsf2 knockdown samples , indicating that biological replicates were similar to each other and different knockdown groups had different expression profiles ( fig . 2c ) . to visualize the distribution of gene expression level for each sample , a boxplot was generated for each of the samples using log10 transformed fpkm values from cufflinks ( fig .
the quartiles and overall range were consistent between biological replicates , indicating that the data were reproducible and of high quality .
alternative splicing events were identified using the dexseq ( v1.10.6 ) package , which models the exon counts as the negative binomial distribution and uses generalized linear models to test differential exon - usage using variance estimated from biological replicates .
sequence depths were used to normalize the exon counts for each sample followed by dispersion estimation before detecting alternative splicing events .
dispersion plots showed that most of the final exon level dispersions follow the fitted line as expected ( fig .
exons with a false discovery rate  <  0.05 , log2 fold change greater than 1.2 , and a minimum of 10 counts for at least one of the samples were considered differentially expressed .
alternative splicing events with chromosome coordinates overlapping with constitutive exons ( ensemble release version 75 ) by at least 1  bp were filtered out using the bioconductor genomicranges ( v 1.18.1 ) package . exons shorter than 12  bp or belonging to differentially expressed genes were filtered out from the list .
dexseq creates a set of disjoint exon fragments ( pseudo exon ) from the ensemble exon annotation file .
here , we described rbfox2 and cpsf2 iclip datasets and their effects on alternative splicing at internal exons and introns .
our results show that cpsf along with sympk is involved in promoting alternative splicing at internal introns and exons and shed light on a new role of mrna 3 end formation factors .
 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 . 
Direct link to deposited data
Experimental design, materials and methods
Cell lines
Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP)
High-throughput sequencing and bioinformatic analysis of iCLIP data
High-throughput sequencing and bioinformatic analysis of RNA-seq data
Discussion
Conflict of interest