RNA-seq analysis of various strains of Mycobacterium tuberculosis.

We have performred RNA-seq analysis of WT Mtb H37Rv, ?phoP Mtb and H37Rv-pde (WT Mtb H37Rv overexpressing gene Rv0805 which codes for a phosphodiesterase), to look for the similarity in ?phoP Mtb and H37Rv-pde transcriptome. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. Briefly, 25 ml of bacterial culture was grown to mid-log phase (OD600= 0.4 to 0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% ß-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer's recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Agrigenome Labs Private Limited (Cochin), India. Overall design: Investigating Mycobacterium tuberculosis transcriptional regulation by PhoP.