RNA-seq of M.smegmatis: w.t. log phase
Cells from 10 mL culture were harvested by centrifugation for 10 min at 3,000  g and 4C, washed twice by 10 ml of fresh Middlebrook 7H9 broth to remove the traces of imidazo[1,2-b][1,2,4,5]tetrazine and DMSO and once by 3 ml of RNAprotect Bacteria Reagent (Qiagen, USA) for RNA stabilization. M. smegmatis cells were homogenized in ExtractRNA reagent (Evrogen, Russia), followed by phenol (pH = 4.5)-chloroform/isoamyl alcohol (25:24:1) purification and precipitation with isopropanol (2:1, v/v). DNase treatment was carried out as previously described with TURBO DNA-free kit (Thermo Fisher Scientific, Waltham, MA, USA), in volumes of 100 L, and further with the RNase-Free DNase Set (Qiagen, Hilden, Germany), according to the manufacturers protocol. RNA cleanup was performed with the RNeasy Mini Kit (Qiagen). The concentration and quality of the total RNA were checked by the Quant-it RiboGreen RNA assay (Thermo Fisher Scientific) and the RNA 6000 Pico chip (Agilent Technologies, Santa Clara, CA, USA), respectively.