the saccharomyces cerevisiae haploid strains used to generate gene expression profiles are listed in table  1 .
deletion strains were obtained from the yeast knockout collection ( yko , open biosystems ) or generated by standard pcr - mediated gene disruption as described , . the set5y402a strain harbors a catalytic inactive set5 protein and was generated as previously described , .
two single colonies for each strain were cultured overnight in ypd media containing 2% dextrose with shaking at 270  rpm at 30  c .
overnight cultures were diluted in 5  ml of ypd to od600  =  0.1 , grown to mid - log phase ( od600  =  0.8 ) shaking at 270  rpm at 30  c .
1.5  ml of each culture were harvested by centrifugation ( 3000  rpm 5  min ) .
pellets were washed in 1  ml of ice - cold water , flash frozen and stored at   80  c until ready to use .
total rna from 1.5  ml of a mid - log culture pellet was isolated using the masterpure yeast rna purification kit ( epicentre ; cat .
the agilent technologies 2100 bioanalyzer instrument was used to assess rna quality and concentration . for all samples ,
, a total of 8  g of purified rna were used as input material for the illumina truseq rna sample preparation v2 low - throughput kit ( illumina ; cat .
samples were processed as specified by the manufacturer 's protocol , using poly - t oligo - attached magnetic beads and two consecutive rounds of enrichment preceding an mrna fragmentation step .
next , fragmented mrna samples were subjected to reverse transcription to generate cdna using superscript ii reverse transcriptase and random primers as indicated by the illumina truseq protocol . the generated cdna was then converted to double stranded cdna and subjected to end repair and 3adenylation .
multiple indexing adapters were then ligated to the end of the ds cdna , followed by a pcr enrichment step .
for all of the resulting libraries , the quality and size  with an expected band approximately at 260  bp  were verified on an agilent technologies 2100 bioanalyzer . indexed libraries from by4741 wildtype , set1 , set5 , set5y402a and set1 set5 mutant strains were subjected to rna - seq on an illumina hiseq2000 platform according to the manufacturer 's protocols ( illumina ) .
the experiment was designed to generate relatively long 101  bp sequences ( single - end ) to improve specificity of the mapping results .
the quality of the raw sequence reads was assessed using fastqc software , with close examination of the  per base sequence quality  results , to ensure accuracy of the base call along the length of the read , and the  overrepresented sequences  results , to ensure the absence of illumina - specific contaminating oligos .
two replicates of each sample were included , with >  10  m mapped reads per replicate .
fastqc did not identify any errors in the quality of the sequenced libraries and no additional read pre - processing steps ( e.g. trimming ) were performed .
gene expression was quantified using the fpkm ( fragments per kilobase of transcript per million mapped reads ) normalization method .
this method estimates the transcript level using the number of reads mapped to a given gene ( read count ) , after normalizing the read count by gene length and the total number of mapped reads in the sample .
we opted to use the tuxedo software suite ( bowtie , tophat , cufflinks , cummerbund ) for fpkm quantification due to ease of installation and usage , as well as integration with the r statistical computing environment . for gene expression quantification ,
specifically , single 101  bp reads were mapped to the s. cerevisiae reference ensembl ef4 genome with tophat , specifying  -no - novel - juncs. gene expression and differential transcription between wt and mutant cells were then determined using cuffdiff .
the cuffdiff program , included in cufflinks , was used to assess biases in read distribution across each transcript and to estimate the statistical significance of gene expression changes between samples .
the cuffdiff test provides q - values , which are p - values adjusted using an optimized false discovery rate ( fdr ) approach .
the cufflinks results were then accessed in the r statistical computing environment using cummerbund ( v2.0.0 ) , and a table of expression values was generated using the fpkmmatrix ( ) function .
the lists of significantly differentially expressed ( sde ) genes were obtained using the getsig ( ) function and are included as text files in the supplementary data .
fpkm expression data is available through geo and included in the supplementary files as  gse52086_processed_data.txt. in addition to the 0.05 q - value threshold from cufflinks , our criteria for defining sde genes included a fold - change threshold of >  1.7 , to ensure that biologically relevant gene expression changes were considered for downstream analyses . using these criteria
, we identified a total of 42 sde genes in set5 cells , 183 sde genes in set1 , and 250 sde genes in set5 set1 cells .
a very similar gene expression profile to set5 was observed for the set5y402a catalytic inactive mutant strain .
set5_sig_genes.txt and  set5set1_sig_genes.txt. after defining sde genes , we explored the characteristics of the mutant genesets by looking at enrichment in specific biological pathways , expression levels , and locations in the genome relative to annotated genomic features . to assess gene set enrichment near certain genomic regions , locations of chromosomal features were downloaded from the saccharomyces genome database ( sgd ) using yeastmine query builder ( included in the supplementary files as 
sgd_genomic_features.tsv ) , and distances from gene transcription start sites ( tss ) to each chromosomal feature were calculated .
significance of enrichment near specific genomic features was tested using the wilcoxon rank sum ( wrs ) statistical method in r. the wrs test is a non - parametric analog to the t test , and enables comparisons of distribution location shift in fpkm expression values from two populations of genes .
the distribution of gene distances to the nearest feature of the indicated geneset was compared with the genome - wide distribution of distances .
reported p values are from the two - sided test , with the alternative hypothesis that the true location shift is not equal to zero .
for the reported significant p values , the geneset distributions were shifted closer to the indicated feature than would be expected by chance given the genome - wide distribution .
epi2014_rnaseq_helper_functions.rdata included in the supplementary files provides the complete set of helper functions used in the rna - seq analyses described above , and supplementary file 
epi2014_set5_dak.r contains the r script used to generate figures 1 , 2 and 3 as in reference .
the saccharomyces cerevisiae haploid strains used to generate gene expression profiles are listed in table  1 .
deletion strains were obtained from the yeast knockout collection ( yko , open biosystems ) or generated by standard pcr - mediated gene disruption as described , . the set5y402a strain harbors a catalytic inactive set5 protein and was generated as previously described , .
two single colonies for each strain were cultured overnight in ypd media containing 2% dextrose with shaking at 270  rpm at 30  c .
overnight cultures were diluted in 5  ml of ypd to od600  =  0.1 , grown to mid - log phase ( od600  =  0.8 ) shaking at 270  rpm at 30  c .
1.5  ml of each culture were harvested by centrifugation ( 3000  rpm 5  min ) .
pellets were washed in 1  ml of ice - cold water , flash frozen and stored at   80  c until ready to use .
total rna from 1.5  ml of a mid - log culture pellet was isolated using the masterpure yeast rna purification kit ( epicentre ; cat .
the agilent technologies 2100 bioanalyzer instrument was used to assess rna quality and concentration . for all samples ,
to enrich for mrna , a total of 8  g of purified rna were used as input material for the illumina truseq rna sample preparation v2 low - throughput kit ( illumina ; cat .
samples were processed as specified by the manufacturer 's protocol , using poly - t oligo - attached magnetic beads and two consecutive rounds of enrichment preceding an mrna fragmentation step .
next , fragmented mrna samples were subjected to reverse transcription to generate cdna using superscript ii reverse transcriptase and random primers as indicated by the illumina truseq protocol .
the generated cdna was then converted to double stranded cdna and subjected to end repair and 3adenylation .
multiple indexing adapters were then ligated to the end of the ds cdna , followed by a pcr enrichment step . for all of the resulting libraries ,
the quality and size  with an expected band approximately at 260  bp  were verified on an agilent technologies 2100 bioanalyzer .
indexed libraries from by4741 wildtype , set1 , set5 , set5y402a and set1 set5 mutant strains were subjected to rna - seq on an illumina hiseq2000 platform according to the manufacturer 's protocols ( illumina ) .
the experiment was designed to generate relatively long 101  bp sequences ( single - end ) to improve specificity of the mapping results .
the quality of the raw sequence reads was assessed using fastqc software , with close examination of the  per base sequence quality  results , to ensure accuracy of the base call along the length of the read , and the  overrepresented sequences  results , to ensure the absence of illumina - specific contaminating oligos .
two replicates of each sample were included , with >  10  m mapped reads per replicate .
fastqc did not identify any errors in the quality of the sequenced libraries and no additional read pre - processing steps ( e.g. trimming ) were performed .
gene expression was quantified using the fpkm ( fragments per kilobase of transcript per million mapped reads ) normalization method .
this method estimates the transcript level using the number of reads mapped to a given gene ( read count ) , after normalizing the read count by gene length and the total number of mapped reads in the sample .
we opted to use the tuxedo software suite ( bowtie , tophat , cufflinks , cummerbund ) for fpkm quantification due to ease of installation and usage , as well as integration with the r statistical computing environment . for gene expression quantification ,
specifically , single 101  bp reads were mapped to the s. cerevisiae reference ensembl ef4 genome with tophat , specifying  -no - novel - juncs. gene expression and differential transcription between wt and mutant cells were then determined using cuffdiff .
the cuffdiff program , included in cufflinks , was used to assess biases in read distribution across each transcript and to estimate the statistical significance of gene expression changes between samples .
the cuffdiff test provides q - values , which are p - values adjusted using an optimized false discovery rate ( fdr ) approach .
the cufflinks results were then accessed in the r statistical computing environment using cummerbund ( v2.0.0 ) , and a table of expression values was generated using the fpkmmatrix ( ) function .
the lists of significantly differentially expressed ( sde ) genes were obtained using the getsig ( ) function and are included as text files in the supplementary data .
fpkm expression data is available through geo and included in the supplementary files as  gse52086_processed_data.txt. in addition to the 0.05 q - value threshold from cufflinks , our criteria for defining sde genes included a fold - change threshold of >  1.7 , to ensure that biologically relevant gene expression changes were considered for downstream analyses . using these criteria
, we identified a total of 42 sde genes in set5 cells , 183 sde genes in set1 , and 250 sde genes in set5 set1 cells .
a very similar gene expression profile to set5 was observed for the set5y402a catalytic inactive mutant strain .
set5_sig_genes.txt and  set5set1_sig_genes.txt. after defining sde genes , we explored the characteristics of the mutant genesets by looking at enrichment in specific biological pathways , expression levels , and locations in the genome relative to annotated genomic features . to assess gene set enrichment near certain genomic regions , locations of chromosomal features were downloaded from the saccharomyces genome database ( sgd ) using yeastmine query builder ( included in the supplementary files as 
sgd_genomic_features.tsv ) , and distances from gene transcription start sites ( tss ) to each chromosomal feature were calculated .
significance of enrichment near specific genomic features was tested using the wilcoxon rank sum ( wrs ) statistical method in r. the wrs test is a non - parametric analog to the t test , and enables comparisons of distribution location shift in fpkm expression values from two populations of genes .
the distribution of gene distances to the nearest feature of the indicated geneset was compared with the genome - wide distribution of distances .
reported p values are from the two - sided test , with the alternative hypothesis that the true location shift is not equal to zero .
for the reported significant p values , the geneset distributions were shifted closer to the indicated feature than would be expected by chance given the genome - wide distribution .
epi2014_rnaseq_helper_functions.rdata included in the supplementary files provides the complete set of helper functions used in the rna - seq analyses described above , and supplementary file 
epi2014_set5_dak.r contains the r script used to generate figures 1 , 2 and 3 as in reference .
this manuscript provides the full complement of experimental and bioinformatics methods developed in ref . , aimed at examining the cooperative functions of the yeast histone methyltransferases set5 and set1 in gene expression .
the r code included here comprises the helper functions used to specifically perform the rna - seq analysis described above (  epi2014_rnaseq_helper_functions.rdata ) , and an annotated script (  epi2014_set5_dak.r ) that outlines in detail the analyses in ref . .
importantly , the script is intended to provide a template for similar gene expression analyses relating transcriptional patterns to chromosomal features with different datasets and model organisms .
overall , this is a useful bioinformatics toolbox that will bring transparency and adaptability to future transcriptome analysis .
 cells regulate transcription by coordinating the activities of multiple histone modifying complexes . 
 we recently identified the yeast histone h4 methyltransferase set5 and discovered functional overlap with the histone h3 methyltransferase set1 in gene expression . specifically , using next - generation rna sequencing ( rna - seq ) , we found that set5 and set1 function synergistically to regulate specific transcriptional programs at subtelomeres and transposable elements . 
 here we provide a comprehensive description of the methodology and analysis tools corresponding to the data deposited in ncbi 's gene expression omnibus ( geo ) under the accession number gse52086 . 
 this data complements the experimental methods described in mas martn g et al . 
 ( 2014 ) and provides the means to explore the cooperative functions of histone h3 and h4 methyltransferases in the regulation of transcription . 
 furthermore , a fully annotated r code is included to enable researchers to use the following computational tools : comparison of significant differential expression ( sde ) profiles ; gene ontology enrichment of sde ; and enrichment of sde relative to chromosomal features , such as centromeres , telomeres , and transposable elements . 
 overall , we present a bioinformatics platform that can be generally implemented for similar analyses with different datasets and in different organisms . 
Experimental design, materials and methods
Yeast strains and media
RNA extraction
RNA-Seq library generation
RNA-Seq and analysis
Discussion
Supplementary data