Transcriptional Profiling of M. tuberculosis

Triplicate M. tuberculosis cultures were grown in modified 7H9 medium to an OD₆₀₀ of 1 and RNA was isolated as previously described using a chloroform-methanol and Trizol (Invitrogen) extraction (Perkowski et al., 2016 (link); Feltcher et al., 2015 (link)). RNA samples were treated with DNase (Promega, Madison, WI) and then column purified (Zymo RNA clean and concentrator Kit, Irvine, CA). Following RNA isolation, cDNA was synthesized with random primers using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was completed using 25 ng of cDNA template in triplicate technical replicates using the SensiMix SYBR and fluorescein kit (Bioline, Toronto, Canada). Transcripts were normalized to the housekeeping gene sigA. Primer sequences are provided in the Key Resources Table.