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four hc and three oligoarticular jia patients were included who all had active disease at the time of sampling .
sf was collected upon therapeutic joint aspiration and pb was drawn at the same moment .
sfmcs and pbmcs were isolated using ficoll isopaque density gradient centrifugation and were frozen down in fcs containing 10% dmso .
cd4cd45ro cells ( ranging between 600.0001.200.000 cells per sample ) were isolated from frozen hc pbmcs and jia sfmcs using flow cytometry , activated with human t - activator cd3/cd28 dynabeads ( 1 cell : 3 beads ) and cultured for 16  h in the presence of 300  nm jq1(+ ) or jq1( ) .
subsequently , cells were treated for 4  h with phorbol 12-myristate 13-acetate ( 100  ng / ml ) and ionomycin ( 1  g / ml ) .
total rna was extracted using the rnaeasy kit ( qiagen ) , according to the manufacturer 's instructions .
the quality of the rna was assessed based on the rna profile generated by the bioanalyzer using the picochip .
removal of rrna and globin - encoding mrna , rna fragmentation , cdna generation , adapter ligation and pcr amplification was done using the truseq stranded total rna with ribo - zero globin sample preparation kit ( illumina ) and 75  bp single - end sequencing was performed using an illumina nextseq 500 platform through the utrecht dna sequencing facility . read quality assessment , sample demultiplexing , and adapter trimming was performed by utrecht dna sequencing facility using basespace ( illumina ) software . reads with quality score of q 
>  30 were selected for downstream analysis . before starting downstream analysis , fastqc ( babraham bioinformatics )
1a and b ) . as a human reference genome , hg19 was used , and reads were mapped to this reference sequence using tophat v.2.0.9   with bowtie 2.1.0   as index ( tophat2 -p 8 library - type fr - firststrand g hg19.gtf ) . for each sample 
  91% of the reads were successfully mapped to the genome , indicating sufficient coverage .
rseqc was used to calculate how mapped reads were distributed over dna regions , such as coding sequences and utrs , and to assess uniform read coverage of genes   ( fig .
transcriptomes of each individual sample were generated using cufflinks v2.2.1 with hg19 gene annotation as a guide , .
quartile normalization was performed ( library - norm quartile ) to improve the detection of differential expression of less abundant transcripts .
reads mapping to rrna and trna were masked ( -m option ) from the quantification .
cuffnorm was used to generate quartile normalized count and fpkm tables . using an fdr    0.05
, 914 transcripts were identified that are significantly differentially expressed between hc and jia patients . among these transcripts
, 637 were upregulated in jia patients compared to hc , while 237 were downregulated in jia patients .
jq1(+ ) and jq1( ) treatments of jia patient - derived memory / effector t ( tmem / eff ) cells resulted in 633 differential transcripts , based on fdr    0.05 , of which 338 were downregulated upon jq1(+ ) treatment and 295 upregulated .
principle component analysis ( pca ) on the complete transcriptome of hc and jia patient - derived t cells , either with and without jq1(+ ) treatment , demonstrated that four different groups can be distinguished , illustrating that hc and jia cluster separately and that jq1 affects gene expression in such way that treated samples cluster separately from untreated samples ( fig .
in addition , we performed pca on the genes significantly expressed between all different groups , which demonstrated that all replicates cluster within the same group ( fig .
initial gene ontology term analysis revealed that the group of significantly genes upregulated in jia compared to hc as well as the genes downregulated upon jq1(+ ) treatment in jia , correlated with a defense response .
additional gene set enrichment analysis ( gsea , broad institute ) for genes differentially expressed between hc and jia showed that the genes positively correlated with this comparison , i.e. genes that are upregulated in jia , are enriched for the same gene subsets as the genes that are negatively correlated with the comparison jq1( ) versus jq1(+ ) treatment of jia patient - derived t cells , i.e. genes that are downregulated by jq1 ( fig .
this again illustrates that genes affected by jq1(+ ) strongly overlap with the genes that are upregulated in jia .
in addition to functionally grouped network analysis , gsea also shows that genes associated with cytokines / cytokine receptor interactions are enriched within this subset of genes . to further validate our rna - sequencing results
, we selected some inflammation - associated ( activation ) markers that were downregulated upon jq1(+ ) treatment and measured gene expression by qpcr .
this resulted in the confirmation of downregulation of several cd markers on the rna level , of which cd38 , cd152 , and cd195 also showed downregulation on the protein level , measured by flow cytometry ( fig .
four hc and three oligoarticular jia patients were included who all had active disease at the time of sampling .
sf was collected upon therapeutic joint aspiration and pb was drawn at the same moment .
sfmcs and pbmcs were isolated using ficoll isopaque density gradient centrifugation and were frozen down in fcs containing 10% dmso .
cd4cd45ro cells ( ranging between 600.0001.200.000 cells per sample ) were isolated from frozen hc pbmcs and jia sfmcs using flow cytometry , activated with human t - activator cd3/cd28 dynabeads ( 1 cell : 3 beads ) and cultured for 16  h in the presence of 300  nm jq1(+ ) or jq1( ) .
subsequently , cells were treated for 4  h with phorbol 12-myristate 13-acetate ( 100  ng / ml ) and ionomycin ( 1  g / ml ) .
total rna was extracted using the rnaeasy kit ( qiagen ) , according to the manufacturer 's instructions .
the quality of the rna was assessed based on the rna profile generated by the bioanalyzer using the picochip .
removal of rrna and globin - encoding mrna , rna fragmentation , cdna generation , adapter ligation and pcr amplification was done using the truseq stranded total rna with ribo - zero globin sample preparation kit ( illumina ) and 75  bp single - end sequencing was performed using an illumina nextseq 500 platform through the utrecht dna sequencing facility .
read quality assessment , sample demultiplexing , and adapter trimming was performed by utrecht dna sequencing facility using basespace ( illumina ) software . reads with quality score of q 
>  30 were selected for downstream analysis . before starting downstream analysis , fastqc ( babraham bioinformatics )
1a and b ) . as a human reference genome , hg19 was used , and reads were mapped to this reference sequence using tophat v.2.0.9   with bowtie 2.1.0   as index ( tophat2 -p 8 library - type fr - firststrand g hg19.gtf ) . for each sample 
  91% of the reads were successfully mapped to the genome , indicating sufficient coverage .
rseqc was used to calculate how mapped reads were distributed over dna regions , such as coding sequences and utrs , and to assess uniform read coverage of genes   ( fig .
transcriptomes of each individual sample were generated using cufflinks v2.2.1 with hg19 gene annotation as a guide , .
quartile normalization was performed ( library - norm quartile ) to improve the detection of differential expression of less abundant transcripts .
reads mapping to rrna and trna were masked ( -m option ) from the quantification .
cuffnorm was used to generate quartile normalized count and fpkm tables . using an fdr    0.05
, 914 transcripts were identified that are significantly differentially expressed between hc and jia patients . among these transcripts ,
637 were upregulated in jia patients compared to hc , while 237 were downregulated in jia patients .
jq1(+ ) and jq1( ) treatments of jia patient - derived memory / effector t ( tmem / eff ) cells resulted in 633 differential transcripts , based on fdr    0.05 , of which 338 were downregulated upon jq1(+ ) treatment and 295 upregulated .
principle component analysis ( pca ) on the complete transcriptome of hc and jia patient - derived t cells , either with and without jq1(+ ) treatment , demonstrated that four different groups can be distinguished , illustrating that hc and jia cluster separately and that jq1 affects gene expression in such way that treated samples cluster separately from untreated samples ( fig .
in addition , we performed pca on the genes significantly expressed between all different groups , which demonstrated that all replicates cluster within the same group ( fig .
initial gene ontology term analysis revealed that the group of significantly genes upregulated in jia compared to hc as well as the genes downregulated upon jq1(+ ) treatment in jia , correlated with a defense response .
additional gene set enrichment analysis ( gsea , broad institute ) for genes differentially expressed between hc and jia showed that the genes positively correlated with this comparison , i.e. genes that are upregulated in jia , are enriched for the same gene subsets as the genes that are negatively correlated with the comparison jq1( ) versus jq1(+ ) treatment of jia patient - derived t cells , i.e. genes that are downregulated by jq1 ( fig . 2c ) .
this again illustrates that genes affected by jq1(+ ) strongly overlap with the genes that are upregulated in jia .
in addition to functionally grouped network analysis , gsea also shows that genes associated with cytokines / cytokine receptor interactions are enriched within this subset of genes . to further validate our rna - sequencing results
, we selected some inflammation - associated ( activation ) markers that were downregulated upon jq1(+ ) treatment and measured gene expression by qpcr .
this resulted in the confirmation of downregulation of several cd markers on the rna level , of which cd38 , cd152 , and cd195 also showed downregulation on the protein level , measured by flow cytometry ( fig .
in order to increase insight into molecular mechanisms playing a role in autoimmune diseases , we performed genome - wide transcriptome profiling of autoinflammatory - site derived tmem / eff cells obtained from jia patients .
we observed that the majority of genes differentially expressed between jia patients and hc were upregulated in jia patient - derived t cells , strongly suggesting that these genes might contribute to the disease .
furthermore , we treated patient - derived t cells with the bet - inhibitor jq1 and observed a preferential inhibition of the genes that were upregulated in jia patients , i.e. the disease - associated genes .
rna - sequencing results were confirmed by qpcr and both pca and gsea showed that jq1 can dramatically alter the cellular gene expression profile . in conclusion ,
our data suggests that bet - inhibition might be a novel , powerful therapeutic strategy in the treatment of autoimmune diseases .
for many autoimmune diseases , the underlying mechanism is still unknown . in order to get more insight into the etiology of autoimmune diseases 
 , we recently published a study were we performed epigenetic profiling and rna sequencing on cd4+cd45ro+ t cells derived from the site of inflammation of juvenile idiopathic arthritis ( jia ) patients and compared this with healthy controls [ 1 ] . in this  data in brief  
 , we focus on the analysis of our rna sequencing data reported in this study , of which the raw and processed files can be found in geo under gse71595 . 
 we provide a detailed description of the downstream analysis , quality controls , and different analysis methods or techniques that validate the results obtained with rna - sequencing .