RNA-seq and genome-wide CRISPRi and Tn-seq analyses of BVL3572S-treated Mycobacterium tuberculosis H37Rv
RNA-seq and genome-wide CRISPRi and Tn-seq analyses of BVL3572S-treated Mycobacterium tuberculosis H37Rv
Cultures (12 mL) were harvested by centrifugation at 3,000 rpm for 10 min. A pooled transposon mutant library of Mtb H37Rv, constructed by transduction with the MycoMar T7 phage (Payros et al. PLoS Pathogens 2021; DOI: 10.1371/journal.ppat.1010020) and containing more than 20,000 mutants, was pre-cultured in 5 mL of 7H9 medium supplemented with Tween 80 0.05%, albumin-dextrose-catalase 10% (ADC, BD Difco) and kanamycin (40 µg/mL) for 5 days at 37 °C. The culture was centrifuged (3,000 rpm, 10 min) and the pellet was washed with 15 mL of Dulbecco's phosphate buffered saline (DPBS 1X, Gibco) with Tween 80 0.05%. Bacteria were resuspended in 25 mL of fresh medium without kanamycin and cultured for 2 days at 37 °C, until an OD600 of approximately 0.4 was reached. The culture was used to inoculate 10 flasks at an OD600 of 0.01 in 25 mL of medium. The flasks were incubated for 3 days at 37 °C until an OD600 nm of 0.2 was reached.  Flasks were separated into 2 groups of 5 and each group was treated with either BVL3572S 27 µM or DMSO 0.8%. After 15 days at 37 °C, 1 mL of each culture was centrifuged (10,000 rpm, 2 min), washed with DPBS 1X supplemented with Tween 80 0.05% and diluted in fresh medium in order to obtain approximately 500,000 bacteria/mL in 25 mL. These cultures were incubated for 7 days at 37 °C up to an OD600 nm of approximately 0.6. Genomic DNA was extracted using the Quick-DNATM Fungal/Bacterial Miniprep Kit (Zymo Research). Transposon-genome junctions were amplified by PCR as described by Payros et al. PLoS Pathogens 2021 (DOI: 10.1371/journal.ppat.1010020). An index specific to each sample was added during this PCR, allowing all samples to be pooled to create the Tn-seq library (TruSeqMC DNA Nano). 