Infections were performed in T75 vented flasks containing monolayers with a confluence of approximately 1×105 cells/cm2. Monolayers were washed 3 times with sterile PBS to remove antibiotics and then 25 ml of fresh medium were added to the monolayer before infection. Inocula for infection were prepared by centrifugation (5000 x g, 15 min) of 10 ml of MAP culture with a density of 8×108 bacteria/ml. Bacterial pellet was resuspended in 10 ml of pre-warmed RPMI medium at 37°C and cells were declumped by 10 passages through a 21 gauge

needle. Monolayers were infected by MAP with a multiplicity of infection (MOI) of 10:1 for 24 h at 37°C at 5% CO2. The next day, extracellular bacteria were killed by amikacin (Sigma) treatment (200 μg/ml) for

2 h at 37°C as already described [24, 25]. Supernatant was removed and monolayer was washed with 3 x PBS rounds. By microscopic examination no extracellular bacteria were detected. buy Go6983 Infected cells were selectively lysed by addiction of 10 ml of lysis buffer per monolayer (4 M guanidine thiocyanate, 0.5% Na N-lauryl sarcosine, 25 mM sodium citrate, and 0.1 M β-mercaptoethanol) without killing intracellular bacteria as previously described [24, 25]. Flasks were shaked at 100 rpm for 15 min at room temperature (RT) and recovered lysate was thoroughly vortexed for 2 min before being passed five times through a 21 gauge needle to shear infected cells and reduce viscosity. One hundred milliliters of lysate belonging to ten T75 flasks were centrifuged at 5000 x g for 30 min at 14°C and pellet was resuspended in 1 ml of fresh lysis buffer. A final centrifugation at 10000 x g for 2 min was performed to harvest bacterial cells AZD6738 order and pellet was then stored at −80°C until RNA extraction. RNA extraction RNA was extracted by using the RiboPure-Bacteria Kit (Ambion) following the manufacturer’s

instructions with some modifications. Briefly, approximately 1×109 mycobacterial cells were resuspended in 350 μl of Adenosine triphosphate RNAWIZ solution (Ambion) and transferred to a 0.5 ml skirted screw-capped microcentrifuge tube containing 300 μl of ice-cold Zirconia Beads. Tubes were immediately processed in the RiboLyser FP120-HY-230 RNA Lysing machine (Hybaid) for three cycles (30 s at speed 6.5) with cooling on ice for 1 min between pulses. Remaining steps were performed according to the manufacturer’s instructions. RNA yield and purity was evaluated with the Nanodrop spectrophotometer (NanoDrop1000, Thermo Scientific) while RNA quality was examined by denaturing gel electrophoresis. All RNA samples were Selleckchem 4SC-202 treated with Dnase I (Ambion) to remove trace amounts of genomic DNA. mRNA enrichment and linear amplification of mycobacterial RNA The 16S and 23S ribosomal RNAs were removed from total RNA (tot-RNA) by using the MICROBExpress Bacterial mRNA Purification Kit (Ambion). Ten micrograms of input tot-RNA were used to get an average of 1–2 μg of output enriched mRNA. rRNAs removal was confirmed by denaturing gel electrophoresis.