design: Batch cultures of Mycolicibacterium sp. ELW1 were grown on minimal salts media (MSM) supplemented with 10 mM fructose for 72 hr (F001-F003). Cultures were split and an aliquot was then washed and collected for RNA-seq. The other aliquot was used to inoculate batch cultures grown in MSM supplemented with 5 mM DEE for 96 hr (D021-D023). Samples were then washed and collected for RNA-seq. Cell pellets were submitted to the North Carolina State Genomic Sciences Laboratory (Raleigh, NC, USA) for RNA extraction, Ribodepletion and Illumina RNA library construction and sequencing. Tissue samples were extracted using a Qiagen RNeasy Total RNA isolation kit and eluted in nuclease-free water. Prior to library construction, RNA integrity, purity, and concentration were assessed using an Agilent Tapestation  RNA assay on the Tapestation 4200 (Agilent Technologies, USA). The samples were depleted using the NEBNext rRNA Depletion Kit for bacteria (NEB). Briefly, the workflow utilizes targeted DNA probes and RNase H to selectively bind to and degrade abundant RNAs. Complementary DNA (cDNA) libraries for Illumina sequencing were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB) and NEBNext Mulitplex Oligos for Illumina (NEB) using the manufacturer-specified protocol. Briefly, the mRNA was chemically fragmented and primed with random oligos for first strand cDNA synthesis. Second strand cDNA synthesis was then carried out with dUTPs to preserve strand orientation information. The double-stranded cDNA was then purified, end repaired and a-tailed for adaptor ligation. Following ligation, the samples were cleaned using AMPure XP bead isolation (Beckman Coulter, USA) with a final library size of 200-600bp. Library enrichment was performed and specific indexes for each sample were added during the protocol-specified PCR amplification. The amplified library fragments were purified and checked for quality and final concentration using an Agilent 4200 Tapestation (DNA Tape, Agilent Technologies, USA). The final quantified libraries were pooled in equimolar amounts for clustering and sequencing on an Illumina NextSeq 2000 DNA sequencer, utilizing a P4 150 PE XLEAP flow cell and sequencing reagent kit (Illumina, USA). The software package Real Time Analysis (RTA), was used to generate raw bcl, or base call files, which were then de-multiplexed by sample into fastq files for data submission.