3.3.1. RNA Extraction

Total RNA was isolated from all *M. tuberculosis* cultures as previously described [32]. In brief, bacteria were re-suspended in 1 mL Trizol (Invitrogen) and added to a 2-mL Lysing Matrix B (MP Bio, USA). Cells were disrupted by bead-beating twice for 1 min with a 2-min interval on ice. The suspension was then transferred to a new tube, where chloroform extraction was performed. RNA was precipitated by adding 0.7 volume of isopropanol and washed with 70% ethanol, air-dried and re-suspended in 100 μL DEPC-treated water.

DNase treatment was carried out with TURBO DNA-free kit (Thermo Fisher Scientific) in volumes of 100 μL and further with the RNase-Free DNase Set (Qiagen, Germany) according to the manufacturer's protocol. RNA cleanup was performed with the RNeasy Mini Kit (Qiagen) according to the RNA Cleanup protocol and stored at −70◦C until further use. DNA contamination was evaluated by PCR, using primers for amplification of the IS*6110* fragment (PolyTub, Lytech, Russia). The concentration and quality of the total extracted RNA were checked by using the Quant-it RiboGreen RNA assay (Thermo Fisher Scientific) and the RNA 6000 pico chip (Agilent Technologies), respectively.

### 3.3.2. RNA-seq and Analysis

Total RNA (1 - 2.5 μg) was used for library preparation. Ribosomal RNA was removed from the total RNA and libraries were prepared using the ScriptSeq Complete kit (Epicentre/Illumina, Madison, USA), according to the manufacturer's protocol. Subsequently, RNA cleanup was performed with the Agencourt RNA Clean XP kit (Beckman Coulter, Brea, USA). The library underwent a final cleanup using the Agencourt AMPure XP system (Beckman Coulter) after which the libraries' size distribution and quality were assessed using a high sensitivity DNA chip (Agilent Technologies). Libraries were subsequently quantified by Quant-iT DNA Assay Kit, High Sensitivity (Thermo Fisher Scientific). Finally, equimolar quantities of all libraries (12 pM) were sequenced by a high throughput run on the Illumina HiSeq using 2×125 bp paired-end reads and a 5% Phix spike-in control. Before loading the cBot system, the libraries were incubated at 98◦C for 2 minutes and then cooled on ice to improve the hybridization of the GC-rich sequences. In total, 114 and 110 million paired reads were obtained corresponding to 14 and 13 billion nucleotide bases for H37Rv and RUS\_B0, respectively. The dataset of RNA-Seq analysis was deposited to the NCBI with the project name PRJNA421323.

Adaptors were trimmed with the Trimmomatic v0.33 tool [45]. Quality control on raw reads was carried out with FASTQC v0.11.5 [46]. The Kallisto v0.46.0 [47] software was used for the reads mapping and abundance estimation. Differential expression analysis was performed using edgeR v3.26.8 [48] package, integrated in the Degust v4.1.1 [49] web-tool. Only genes with count per million (CPM) ≥ 1 were analyzed further. Genes were filtered based on false discovery rate cutoff (FDR) ≤ 0.05 and minimum expression fold change (FC) ≥ 2. The plots were generated using the ggplot2 package in R.
