Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Jmjd6-/- Thymic stromal Transcriptomes

The goals of this study are to comprehensively identify genes controlled by Jmjd6 in the thymic stroma, and to identify a novel alternative splicing mechanism. Methods: Samples were WT and Jmjd6-/- fetal thymus organ culture with (2 samples for each category) or without (1 sample for each category) RANKL stimulation for 4 days under 2-DG. One µg of total RNA was used for library construction with TruSeq RNA Sample Prep Kit v2. THe ligated products were amplified using 8 cycles of PCR to generate RNA-seq library. Library integrity was verified by Bioanalyzer DNA1000 assay. Sequencing was performed in 101-bp paired-end mode using an Illumina HiSeq.Technical duplicate has done. Results: A total of 177,060,020 reads were obtained for 6 samples. Filtered reads were mapped to the UCSC mm10 using the TopHat program(v2.0.10) with the default parameters. The Cufflinks program (v2.1.1) was then used to assemble 22,448 transcripts and to calculate the fragments per kilobase of exon per million mapped fragments(FPKM) values, which are normalized measurement of gene expression levels(= genes-FPKM file).To identify differentially expressed genes, the ratio of the maximum FPKM to the minimum FPKM was compared among 6 samples. When the ratio was more than 3, the gene was regarded as being significantly altered in expression level. We added 0.1 to the FPKM value to avoid division by zero. This led us to identify 3212 genes with differential expression. Among these, the expression levels of 2536 genes were significantly associated with the RANKL treatment or Jmjd6 expression ( P value <0.05). Analysis for intron retention was performed as follows. According to the current gene annotation ("known genes" in UCSC mm10), there are 188,208 introns in total. As intron retention events should be observed in the genes with relatively high expression, we only focused on the genes with the maximum FPKM value more than 10 at least in one of the six samples. As a result, we obtained 84,708 introns. The reads mapped to these intronic regions were counted by the intersectBed program in the BEDTools utilities with -c option, and the counts are converted into the FPKM values for each intron (intronic FPKM). There are 1051 introns with intronic FPKM more than 10 in at last on of the six samples, and the degree of intron retention was calculated by dividing intronic FPKM value by conventional FPKM value for each gene (intron-FPKM file). Overall design: Samples were WT and Jmjd6-/- fetal thymus organ culture with (2 samples for each category) or without (1 sample for each category) RANKL stimulation for 4 days under 2-DG. One µg of total RNA was used for library construction with TruSeq RNA Sample Prep Kit v2. THe ligated products were amplified using 8 cycles of PCR to generate RNA-seq library. Library integrity was verified by Bioanalyzer DNA1000 assay. Sequencing was performed in 101-bp paired-end mode using an Illumina HiSeq.Technical duplicate has done.

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Yanagihara T, Sanematsu F, Sato T, Uruno T, Duan X, Tomino T, Harada Y, Watanabe M, Wang Y, Tanaka Y, Nakanishi Y, Suyama M, Yoshinori F