title: GSM7830790: Ai2: Mtb, A-ALF, after infection, rep2; Mycobacterium tuberculosis; RNA-Seq
library_construction_protocol: Bacterial RNA was extracted using the Quick-RNA Fungal/Bacterial Miniprep kit (Zymo Research). Briefly, bacterial pellets were suspended in RNA lysis buffer and transferred to a ZR BashingBead Lysis tube containing 0.1 mm and 0.5 mm ceramic beads. Samples underwent a bead beating procedure in a Disruptor Genie (10 cycles of 1 min at maximum speed with 1 min intervals on ice), facilitating the breaking down of the M.tb cell envelope. RNA in the supernatant was isolated using the Zymo-spin columns, including an in-column DNAse I treatment, and was finally eluted in nuclease-free water. A second DNAse treatment was performed on the extracted RNA using the TURBO DNAse reagent (Thermo Fisher Scientific, Waltham, MA, USA). RNA was further purified using the RNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA),  MA, USA). Conversely, infected ATs (at 72 hpi) were washed twice with culture media and then lysed with GTC solution (4M guanidinium thiocyanate, 0.5% sodium N-lauryl sarcosine, 25 mM tri-sodium citrate, 0.1M 2-mercaptoethanol, 0.5% Tween 80, pH 7.0) 34, 35 for 5 min at RT and then centrifuged at 5000 x g for 30 min at RT to recover bacteria. M.tb pellets were washed in GTC lysis solution to combine the content from all flasks (4 x 150 cm2 flasks/ condition) into one single tube, centrifuged, and suspended in RNA lysis buffer. RNA was then extracted using the Quick-RNA Fungal/Bacterial Miniprep kit, treated with TURBO DNAse, and purified with the RNA Clean & Concentrator kit, as previously described. A whole-stranded transcriptome RNA library was generated using the Zymo-Seq RiboFree Total RNA kit (Zymo Research, Irvine, CA, USA), following the manufacturer's protocol. RNA (250 ng) was used as input to construct the library, which included a rRNA depletion step of 30 min followed by 15 cycles of PCR. Using a NextSeq 500 Mid Output Illumina platform, a 75-bp paired-end (PE) sequencing was conducted. RNA sequencing was performed on three independent experiments (n = 3 per condition), except for E-ALF-exposed M.tb before infection (n=2), where one of the replicates was not included in the study due to low RNA concentration and quality.