Expression profiling of Mycobacterium smegmatis in M63 medium and M63 medium supplemented with NH4Cl for wild-type, eccCa1, eccD, esxB

Mycobacterium smegmatis respond to various environmental stimuli via ESX systems. We probed the transcriptional response to nitrogen addition to the medium using a series of ESX-1 knockout mutants. Overall design: Expression profiling of Mycobacterium smegmatis in M63 medium and M63 medium supplemented with NH4Cl for wild-type and eccCa1, eccD, esxB mutants RNA was extracted from cultures grown to OD600 nm ~ 0.7-0.8. Three biological replicates (single colonies) were prepared for every strain assayed. The NEB RNA extraction kit was used as directed except for the lysis step, which was completed by bead beating the cells with 0.1 mm zirconia beads. To deplete rRNA, we used the NEB rRNA kit as directed. Samples were prepared using the NEBNext Ultra RNA library prep kit for Illumina, and barcoding oligos were used to pool the libraries. Two technical replicates were prepared for analysis. The resulting pools were sequenced with the Illumina NovaSeq in 75 nt single end reads, resulting in 15M reads per sample.