GSM5342055: MED, mtBCG cultured at exponential phase in dextrose; Mycobacterium tuberculosis variant bovis BCG; RNA-Seq
Total RNA from each culture condition was isolated as previously reported (Soto-Ramirez et al., 2017) and purified using Trizol™ reagent (Invitrogen, USA). As two strains (wtBCG and mtBCG) were used and four conditions were tested (exponential, stationary, dextrose and lipids). All samples  passed the quality tests required for RNAseq analysis: 1) concentration was evaluated using Qubit™ RNA HS Assay Kit in the Qubit™ 2.0 fluorometer, 2) DNA absence was analyzed by qPCR and 3) RNA integrity was analyzed with a bioanalyzer (Agilent Technologies). RNA-seq libraries were prepared in accordance with the protocols reported previously (Rodriguez et al., 2014; Waldbauer et al.,2012). Briefly, RNA samples were diluted at a concentration of 0.4 µg/µL for rRNA removal using the mRNA Enrichment, MICROBExpress™ kit (Invitrogen, USA). mRNA was precipitated and solubilized in 20 µL DEPC-treated water. Purified mRNA was fragmented by divalent-cation hydrolysis (Fragmentation Buffer; Ambion, Austin, TX) at 70°C for 12 min to yield fragment sizes between 60 and 200 nt. After purification by the RNA Clean and Concentrator™ kit (Zymo Research, USA), mRNA samples were subjected to poly(A) tailing and end repairing (NEB Reagents). RNA was treated with antarctic phosphatase (New England Biolabs) and then phosphorylated at the 5' ends with T4 PNK (New England Biolabs). The transcribed strand was labeled by ligation of a 5' hybrid DNA-RNA primer (5-hybrid-0) and after purification with RNA clean XP beads (Beckman Coulter Genomics), the first strand cDNA synthesis reaction was carried out with SuperScript II reverse transcriptase (Invitrogen, USA) and Illumina's poly(T) primer (IGA-dT16-VN). Reaction components of cDNA synthesis were removed by treatment with Agencourt AMPure XP SPRI beads (Beckman, USA), and primary transcripts were enriched with a high-fidelity polymerase (Accuzyme™ kit, Bioline) and the Illumina spacers as primers. Illumina adaptors and bar codes were ligated by PCR in accordance with the manufacturer's instructions. Libraries were purified with SPRI beads (Beckman) and evaluated by Qubit™ 2.0 and Bioanalyzer (Agilent Technologies).  Finally, we obtained cDNA libraries with size of 350 bp and concentrations from 12.5 ng/µL to 35.7 ng/µL
GEO
GSM5342055