Mycobacterial RNA Isolation and qRT-PCR Analysis

The RNA from Mtb strains was isolated as described in Sharma et al. (2019 (link)). Approximately 2 × 10⁹ WT and KO mycobacterial cells were used for RNA extraction. The RNA was isolated from the WT and KO pellets with a DNA, RNA, and protein purification kit (Machery-Nagel NucleoSpin^TM; Germany) as per the manufacturer's protocol. Three μg of the eluted RNA were treated with 1 μl of Turbo DNase enzyme (Turbo DNA-free Kit; Thermo Fischer Scientific, United States) to avoid contaminating the genomic DNA. Two μg of the DNase-treated RNA was used as template to generate cDNA as per PrimeScript 1st strand cDNA synthesis kit (Takara, Japan). One μl of the generated cDNA for each sample was taken for quantitative real-time PCR (qRT-PCR) using 5 × HOT FIREPol Evagreen qPCR Mix Plus (SYBR Green; Solis Biodyne, Estonia) on the Stratagene mx3005p system (Agilent Technologies, United States). The primer pairs used are indicated in Supplementary Table 3. sigA transcript levels (Ct value) in different strains were used as internal controls for normalization and accurate estimation of Ct values of all genes under study.