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. rRNA were depleted using an in-house enzyme and poly(A)-tailed. 5' Illumina TruSeq sequencing adapter, carrying sequences of ATTACTCG and TCGGAGA (50% each) were ligated to 5' monophosphorylated RNA. Total RNA was then treated with Terminator RNA exonuclease (Lucigen) following the manufacturer's recommendations to remove unligated 5' monophosphorylated RNAs. Conversion of Triphosphates to monophosphastes groups was carried by the 5' polyphosphatase (Epicentre) using manufacturer's protocol and distinct 5' Illumina sequencing adapters carrying sequences of CGCTCATT and GAGATTCC (50% of each) were ligated to monophosphorylated RNAs before fragmentation. DNA-free total RNA were fragmented with ultrasounds 4 pulses of 30 sec at 4°C and library were then constructed with the TruSeq RNA library Kit V2 (Illumina).