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
10 mL of the treated and control cultures were harvested by centrifugation at 3,000 x g for 5 min. 25 mL cultures were grown at 37°C without agitation to an OD600 of 0.7. Cultures were incubated with BVL3572S 20 µM or with DMSO 0.5% for 6 h at 37°C without agitation. The pellets were resuspended in 1 mL of RNApro (FastRNA Pro Blue Kit, MP biomedicals) and homogenized in impact-resistant 2 mL tubes containing 0.1 mm silica spheres (Lysing Matrix B, MP biomedicals) using a FastPrep FP120 cell disrupter (Thermo Fisher Scientific) at 6.0 Hz for 40 s. The lysates were centrifuged at 12,000 x g and RNA was purified according to the manufacturer's instructions. Ribosomal RNA (rRNA) was depleted using the QIAseq FastSelect 5S/16S/23S kit (Qiagen).  Illumina sequencing libraires were prepared with the TruSeq RNA sample preparation kit version 2.0 rev. A (Illumina Inc.) with a unique index for each cDNA library.