Dnase 1

Total RNA was extracted from cells using RNeasy Protect Cell Mini Kit (Qiagen) with QIAshredder homogenizers (Qiagen). RNA was treated with DNase I (PrimerDesign or Invitrogen) according to manufacturer’s instruction, samples’ concentration was adjusted to 50 ng/µl and verified on Qubit Fluorometer (Invitrogen) using Qubit RNA HS Assay Kit (Invitrogen). Expression of DHRS9 and Ido1 mRNA was measured by RT-qPCR relative to GAPDH mRNA endogenous control using TaqPath 1-Step Multiplex Master Mix Kit (Applied Biosystems) and QuantStudio 5 Real-time PCR system (Applied Biosystems) according to manufacturer’s instruction. TaqMan assays were from Applied Biosystems: for Human Ido-1 assay number Hs00984148_m1 (FAM-MGB), for Human DHRS9 Hs00608375_m1 (FAM-MGB) and for Human GAPDH Hs03929097_g1 (VIC-MGB). Each amplification reaction contained 50 ng of RNA and was performed in triplicates. Data was analyzed with QuantStudio Design and Analysis desktop Software (Applied Biosystems). Changes in expression (Rq) were calculated relative to expression of corresponding mRNA in the starting material, i.e. CD14 + monocytes.

Mycobacteria were recovered using a guanidine thiocyanate (GTC)/Trizol extraction method as previously described^{48 (link)}. RNA was extracted by bead-beating with 0.1 mm silica beads for 45 s at speed 6.5 m/s (MP Biomedicals, Santa Ana, USA), followed by chloroform extraction and RNA purification using the mirVana RNA isolation kit (ThermoFisher Scientific). Mycobacterial RNA was treated with DNase I (Primerdesign, Southampton, UK), and RNA yield and quality assessed using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies) and Agilent 2100 Bioanalyzer (Agilent Technologies). RNA samples were directly labelled with Cy3 fluorophore using the Universal Linkage System (ULS, Kreatech Diagnostics), as previously described^{49 (link)}, and hybridised to high density Agilent tiling arrays with 180,000 60-mer oligonucleotides evenly tiled across the M. tb H37Rv genome, designed by the Bacterial Microarray Group at St George’s, University of London (ArrayExpress accession A-BUGS-47). Expression ratios were generated by averaging the antisense probes for each gene in the M. bovis BCG Pasteur genome. RNA from three independent biological replicates of RCCS-biofilm were contrasted to log phase (day 5; OD 0.400 ± 0.053, ~7 × 10⁶ CFU/mL) and stationary phase (day 21, OD 0.580 ± 0.040, ~2 × 10⁸ CFU/mL) bacilli, cultured in triplicate in static Sauton media no Tween 80.