Characterize the regulon of 6C sRNA in Mycobacteria smegmatis

Purpose: The goals of this study are to identify the putative mRNA targets that are regulated by the 6C sRNA. We constuct an inducible vector to transiently overexpressed the 6C sRNA in M. smegmatis, and then we perform RNA-Seq to look for genes that are differicently expressed upon the over-expression of 6C sRNA, which we think these genes are the potential targets of the 6C sRNA. Overall design: Methods: Purified RNA was used to construct cDNA library according to the TruSeq Stranded RNA LT Guide from Illumina. High-throughput sequencing was carried out on an Illumina HiSeq 2000 system according to the manufacturer's instructions (Illumina HiSeq 2000 User Guide) and 150-bp paired-end reads were obtained. The raw reads were filtered by Seqtk and then mapped to the M. smegmatis MC2 155 strain reference sequence (GenBank NC_008596) using Bowtie2 (version: 2-2.0.5). Counting of reads per gene was performed using HTSeq followed by TMM (trimmed mean of M-values) normalization. Differentially expressed genes were defined as those with a false discovery rate < 0.05 and fold-change >2 using the edgeR software.