GSM6068953: H37Rv WT - rep 1; Mycobacterium tuberculosis; RNA-Seq
bacteria were harvested by centrifugation and the pellet washed in GTC buffer (4 M guanidine thiocyanate, 0.5% Na N-lauryl sarcosine, 25 mM Na citrate, pH 7.0, 0.1 M beta-mercaptoethanol) followed by phosphate-buffered saline (PBS)–0.1% Tween 80. Bacteria were disrupted by treatment with lysozyme followed by bead beating in the presence of warm TRIzol. RNA was subsequently isolated using DirectZol RNA isolation columns (Zymo). RNA was eluted in 50 L RNase-free water and then treated with DNase (Ambion) to ensure removal of genomic DNA contamination. Starting with total RNA, libraries were prepared according to Illumina instructions. Ribosomal RNA was removed using Ribo-Zero rRNA. Stranded RNA Library Prep was performed on a Wafergen Bio-systems Apollo 324 Robot. Libraries were quantified by qPCR.