GSM5958517: M. tuberculosis Beijing-Like 323 Day 3 Post Infection Biological Replicate 2 - Technical Replicate 3; Mycobacterium tuberculosis; RNA-Seq
Lungs stored at -80°C were removed and maintained in liquid nitrogen until processing. All the procedures were handled at 4ºC to avoid RNA degradation. Both mouse lungs were pulverized with liquid nitrogen, and aliquots of lung powder were stored to -80°C until used. To release the bacterial cells, mild-lysis buffer RLT Plus (Cat No: 1053393 Qiagen) supplemented with 10% β-mercaptoethanol was added. After centrifugation at 14,000 rpm/4ºC for 5 min, the bacterial enriched pellet was used for RNA extraction using the Quick-RNA Miniprep Kit (Zymo Research), following the manufacturer's instructions Depletion of eukaryotic ribosomal subunits was carried out with the Ribo-Zero Kit (llumina® Ribo-Zero Plus rRNA Depletion Kit Cat: 20037135) following the manufacturer's instructions. Depleted RNA (500 ng of each sample) was used to construct libraries using the TruSeq® Stranded mRNA llumina® kit. The fragmentation time was adjusted to 14 min to obtain ~ 200-400 bp inserts, and the PCR enrichment was set to 15 cycles. To ensure good quality and quantity for the libraries, the concentration was verified by Qubit™ dsDNA HS (ThermoFisher Cat: Q32852), and the size of the libraries was verified using Agilent 2100 bioanalyzer System High Sensitivity DNA Kit (Cat: 5067-4626). The libraries with the correct insert size were subjected to bacterial ribosomal subunits depletion with an in-house subtractive hybridization TruSeq® Stranded mRNA llumina®