NextSeq 500 sequencing; RNA-seq, term-seq and tagRNA-seq of M. tuberculosis strains H37Rv and RhoDUC strains induced for Rho depletion at times 0H, 1.5H, 3H, 4.5H and 6H
Mycobacterium tuberculosis H37Rv and RhoDUC were cultured in liquid Middlebrook 7H9 supplemented with 10% ADC (Sigma), 0.5% Glycerol and 50 µg/ml hygromycin if appropriate and 0.05% Tween 80. Cultures were harvested at an OD600nm ~ 0.6 for exponential phase. Cells were stopped using 30% ice, centrifuged at 5000rpm for 10min at 4°C and the RNA extracted using the FastRNA Pro Blue Kit (MP Biomedicals) following the instruction of the supplier. RNA concentration and purity was assessed using Nanodrop 2000 (ThermoFisher). RNA integrity was assessed using 2100 Bioanalyzer (Agilent) for RNA sequencing. Total rRNA were depleted using an in-house enzyme and the 5' Illumina adenylated sequencing adapter was ligated to the 3'end of the total RNA. First-strand cDNA was reverse transcribed using the M-MLV reverse transcriptase before fragmentation and library construction. DNA-free total RNA were fragmented with ultrasounds 4 pulses of 30 sec at 4°C and the library was constructed with the TruSeq RNA library Kit V2 (Illumina).