GSM9726611: Mouse sample 2 rep 1; Mycobacterium tuberculosis; Mus musculus; RNA-Seq
Bacterial RNA was harvested from infected macrophages as previously described (Pisu et al., 2020). Infected macrophages were lysed in TRIzol (Thermo Fisher Scientific) and centrifuged at 10,000xg for 20 min at 4 °C to pellet intact bacteria. The supernatant, representing the host RNA fraction, was collected and filtered twice through a 0.2 µm PVDF filter to ensure pathogen inactivation. The bacterial pellet was resuspended in fresh TRIzol containing Lysing Matrix B beads (MP Biomedicals) and disrupted using a bead beater homogenizer (MP Biomedical) at 6.5 m/s for 45s, followed by a 5 min incubation on ice and a second 30 s bead-beating cycle. Following lysis, the host and bacterial RNA fractions were combined, then extracted with chloroform (200 µL per 1 mL TRIzol), and purified using on-column cleanup with DNase treatment (RNA Clean & Concentrate, Zymo Research). 200ng of total RNA was used as input for strand-specific library preparation with the Agilent SureSelect XT HS2 RNA Kit, following the manufacturer's instructions, with a 1:2 adapter dilution, and an additional 0.8X/1.2X double SPRI post-ligation to deplete ribosomal RNA. For intracellular bacterial RNA sequencing, 200ng unpooled libraries were hybrid-selected for bacterial transcripts using a custom set of biotinylated probes complementary to the Mtb H37Rv transcriptome, excluding ribosomal and transfer RNAs, and probes were used at a 1:5 dilution to account for bacterial transcript being a low proportion of input libraries. All libraries were sequenced on an Illumina NextSeq 550 using 150 bp reads.