title: GSM5468245: WT Rep 2, MOPS buffer; Mycolicibacterium smegmatis MC2 155; RNA-Seq
library_construction_protocol: RNA was extracted using the method Rohde, Abramovitch and Russell, Cell Host and Microbe, 2007. Briefly, RNA was stabilized and isolated using a guanidine thiocyanate-based lysis buffer as previously described. Pelleted mycobacteria were lysed using 5 μg/ml lysozyme, hot Trizol, and physical disruption with silica beads in a BeadBeater. Total RNA was isolated by chloroform extraction followed by direct application to QIAGEN RNeasy column purification. Residual DNA contamination was removed using Turbo DNAfree DNase (Ambion). RNA quality and integrity were examined using an Agilent Bioanalyzer prior to subjecting samples to rRNA depletion using the Epicentre Ribo-Zero depletion kit. cDNA libraries were constructed using the Illumina TruSeq RNA library preparation kit (v2), omitting the polyA selection step. After library quality control, sample libraries were pooled and sequenced in one lane of an Illumina HiSeq 2500 Rapid Run flow cell (v1). After the sequencing run, reads were de-multiplexed and converted to FASTQ format using the Illumina bcl2fastq (v1.8.4) script.