title: NextSeq 500 paired end sequencing; Dissecting the Mycobacterium bovis BCG response to macrophage infection to identify targets for anti-tuberculosis drug and vaccine discovery
design: Dissecting the Mycobacterium bovis BCG response to macrophage infection to identify targets for anti-tuberculosis drug and vaccine discovery
library_construction_protocol: Mycobacterium bovis BCG Montreal (ATCC 35735), containing pEGFP cloned under the control of mycobacterial 19 kDa promoter (Bettencourt et al., 2020), was sub-cultured from frozen in Middlebrook 7H9 medium (Sigma, MO, USA) with 0.05% v/v tyloxapol supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.5% glycerol. Log phase bacteria for macrophage infections were harvested from 50 mL cultures in 250 mL vented Erlenmeyer flasks (Corning, NY, USA) after three days incubation at 37C with shaking (180 rpm). Log phase M.bovis BCG RNA was extracted after five-day axenic culture (OD 0.35-0.46) from three independent biological replicates. Human monocyte THP-1 cells (ATCC:TIB-202) were maintained at 37C, 5% CO2 in RPMI medium supplemented with 2 mM L-glutamine, 10% v/v fetal bovine serum and 1 mM sodium pyruvate without antibiotics. THP-1 cells (1.1 x 106 cells/mL) were differentiated into macrophage-like cells by 24h stimulation with phorbol 12-myristate 13-acetate (20 nM final concentration). After washing twice with phosphate-buffered saline (PBS), cells were rested for 48h in RPMI before infection. M.bovis BCG harvested from log phase cultures were washed in PBS, resuspended in RPMI media, and syringed five times to generate a homogenous cell suspension before infecting macrophages at a multiplicity of infection of 10 bacilli: 1 macrophage for 4h. After infection, cells were washed with sterile PBS to remove extracellular bacteria before incubation with fresh RPMI for 20h at 37C in a 5% CO2 humidified incubator (Bettencourt et al., 2020). RNA was extracted from intracellular mycobacteria and from in vitro log phase M.bovis BCG using the GTC/TRIzol differential lysis method (Tailleux et al., 2008). Mycobacterial RNA was DNase-treated and purified using the RNeasy spin-column system (Qiagen, MD, USA). RNA quality and yield was evaluated using the Qubit Broad Range RNA assay (Thermo Scientific, MA, USA), Nanodrop One (Thermo Scientific, MA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Three independent biological replicates of intracellular M.bovis BCG (2 g/replicate) and 3 replicates of in vitro log phase M.bovis BCG RNA (2 g/replicate) were depleted of mycobacterial rRNA using Ribo-Zero rRNA Removal (Bacteria) kit (Illumina, CA, USA). Sequencing libraries were prepared using the NEBNext Ultra II kit (New England Biolabs, MA, USA) and paired-end sequenced using the Illumina NextSeq500 (75 x 2 paired end) instrument.