NextSeq 500 paired end sequencing; Identification of antigens presented by MHC for vaccines against tuberculosis
Identification of antigens presented by MHC for vaccines against tuberculosis
Human monocyte THP-1 cells were differentiated into macrophage-like cells with 200 nM PMA for 24h, followed by 48h recovery in PMA-free media. Exponential phase M. bovis BCG Pasteur-GFP  was washed in phosphate-buffered saline and re-suspended in RPMI without antibiotics. The inoculum was needle homogenised fifteen times to create a single-cell suspension. THP-1 cells were infected at an MOI of 10:1 for 4h at 37ºC with 5% CO2. After 4h, the cells were washed with PBS and then incubated in RPMI without antibiotics for a further 20h. Mycobacterial RNA was harvested using the GTC/Trizol differential lysis method and purified using RNeasy columns (Qiagen) with DNase treatment. Mycobacterial RNA yield and quality was assessed using the NanoDrop One Spectrophotometer (Thermo Scientific) and Agilent 4200 TapeStation (Agilent Technologies). Libraries for RNA sequencing were prepared from three (2ug total RNA) biological replicates of intracellular M. bovis BCG after rRNA depletion (Ribo-Zero, Illumina) using the NEBNext Ultra II kits (New England Biolabs), and sequenced on an Illumina NextSeq500 sequencer.
Brighton and Sussex Medical School, University of Sussex, UK
E-MTAB-8057:Intracellular BCG 5_2_B_p