Transcriptomic Analysis of Mtb-Infected Monocytes

Cryopreserved PBMCs (n=115) were thawed and resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 50 ng/mL recombinant human monocyte colony-stimulated factor (M-CSF) for 24h. CD14⁺ monocytes were enriched from PBMCs by magnetic separation (Monocyte Isolation Kit II, Miltenyi Biotec) and incubated in RPMI-10 with M-CSF for 24h. Monocytes were then infected with H37Rv Mtb at a multiplicity of infection (MOI) of 1 or media alone (uninfected control) and incubated at 37°C with 5% CO² for 6h. After incubation, both the media-only and Mtb-infected monocytes were lysed in TRIzol (Invitrogen), and RNA was isolated using the RNeasy Mini Kit according to the manufacturers’ instructions (Qiagen). The RNA samples were quantified using Nanodrop 8000 instrument (Thermo Scientific), and their quality was measured by TapeStation (Agilent) (RNA Integrity Number ≥ 8.0). Next, cDNA libraries were prepared with random hexanucleotide primers and rRNA depletion using SMARTer RNAseq Kit (Takara), followed by sequencing on Illumina HiSeq 2500 and Novaseq 6000 platforms as previously described (15 (link)).