RNA-seq of peritoneal macrophages transfected by si-Tet2 with or without LM or IFN-? treatment

Purpose:The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: total mRNA profiles of si-NCand si-Tet2 transfected macrophages were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow,we knocked down Tet2 in mouse PMs and infected them with LM. RNA sequencing data revealed a broad spectrum of changes in the expression of many cytokines and immune-related genes upon LM infection. Downregulation of Tet2 drastically reversed this LM-induced cytokine overexpression at 12 h post-infection Conclusions:This finding indicates that Tet2 is either required for cytokine production in immune response, or conversely, Tet2 protects bacteria from clearance. Ablation of Tet2 may accelerate the bacterial clearance in host macrophages, in which cytokines are not needed at the late time point. Overall design: RNA-seq detection of PMs with related treatments