Transcriptomic analysis of Mtb under K+ deprivation

Log-phase (OD₆₀₀ ~ 0.6) Mtb was used to inoculate standing, vented, T-25 flasks containing either 10 ml of standard 7H9, pH 7 medium ([K⁺] = 7.35 mM) or K⁺-free 7H9, pH 7 medium at OD₆₀₀ = 0.3. K⁺-free 7H9 was made by replacing monopotassium phosphate with monosodium phosphate. To make Na⁺-free 7H9, disodium phosphate was replaced with dipotassium phosphate, monosodium glutamate with monopotassium glutamate, and sodium citrate with potassium citrate. The media was supplemented with 0.5% bovine serum albumin, 0.2% dextrose, and 14.5 mM KCl, and brought to pH 7 with KOH. Samples were collected 4 hours post-exposure and RNA isolation carried out as previously described [8 (link)]. Two biological replicates per condition were used for RNA sequencing. Library preparation using Ribo-Zero rRNA removal (bacterial) and TruSeq Stranded kits (Illumina) were performed by the Tufts University Genomics Core Facility. Barcoded samples were pooled and run on a single lane on an Illumina HiSeq 2500 (High Output v4) with single-end 100 bp reads. Data were analyzed using the SPARTA program [64 (link)]. qRT-PCR experiments were performed as previously described, except cDNA was synthesized from 250 ng of RNA without prior amplification [2 (link), 37 (link)].