GSM7814645: phoR KO 7.0-REPLICATE 3; Mycobacterium tuberculosis; RNA-Seq
RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. Briefly, 25 ml of bacterial culture was grown (in the indicated pH) to mid-log phase (OD600= 0.4 to 0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-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). Total RNA was isolated using Direct-zol RNA MiniPrep kit(Zymo Research Corporation),thepurity of isolated RNA was determined using the NanoDrop (ThermoScientific), quantified using Qubit (Invitrogen), and 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.Briefly,The rRNA was removed using 'si-tools Pan-prokaryote ribopool probes,' and further library preparation was carried out using TruSeq Stranded RNA library prep kit. Prepared libraries were sequenced using Illumina HiSeq x10 platform, and 150-bp paired-end reads were generated.Thereads that passed quality control were mapped onto the reference genome ofM. tuberculosisH37Rv usingHisat2(version-2.0.5).The abundance of transcripts in individual samples was estimated usingCufflinks (v2.2.1).