RASA3 regulates the balance of pTh17-Th2 reciprocal programs.

Total RNAof pTh17 cells from wild type (WT) and RASA3 KO (RASA3KO) mouse (C57BL/6) was extracted and RNA-seq libraries were generated. Reads (32-45 Million reads per sample) were analyzed with Salmon software to align and quantify the transcript expression. R packages in Bioconductor, tximport and tximportData were used to aggregate transcript-level quantifications to the gene level, with the R package biomaRt for gene and transcripts mapping. The option "lengthScaledTPM" for countsFromAbundance in tximport was used to obtain the estimated counts at the gene level using abundance estimates scaled based on the average transcript length over samples and the library size. R function voom in limma package was used to transform the estimated count data into log2 scale and estimate the mean-variance relationship so that it can be used to compute appropriate observation level weights, followed by linear modelling. For gene-level differential expression analysis, a linear model was fitted to the log scaled expression data with the genotypes (knockout and wild-type) as one covariate using empirical Bayes moderated t-statistics. The false discovery rate (FDR) was controlled using the Benjamini and Hochberg algorithm. Probes with FDR < 0.05 and fold-change > 2 were judged to be differentially expressed. 317 and 35 genes were identified to be significantly up- and down-regulated respectively. The R function CAMERA was used to determine whether each gene set was differentially expressed in the comparisons as a set. The differential expression patterns and to calculate the enrichment scores of Th1/Th2 (KEGG mmu04658) and Th17 (KEGG mmu04659) related genes in the knockout vs. wild-type comparisons. Our analysis demonstrated that increase of Th2 program and decrease Th17 program in RASA3 KO pTh17 cells, with no change of Th1 related genes. This study indicated that RASA3 is vital for the balance of pTh17-Th2 reciprocal programs. Examination of differential gene expression in WT and RASA3 KO pTh17 cells at 6hr after differentiatoin.