GSM8253966: UP014,HC,Uninfected,ScRNAseq; Homo sapiens; RNA-Seq
Fresh BAL fluid was filtered through a 70-um strainer into a 50 ml falcon conical tube to remove any clumped cells and mucus. Pelleted BAL cells were isolated by centrifugation at 1500 rpm for 5 min at 40∘C and supernatant removed and stored. BAL cells were washed twice in 1x DPBS (Gibco) and re-suspended in complete RPMI 1640 media with L-glutamine, 10%  fetal bovine serum (Gibco), and penicillin-streptomycin (Gibco). BAL cells were infected with Mtb H37Rv at an MOI of 2:1 (bacteria: cells) and only media was added to control wells. For optimal contact, the cells in the plate were further centrifuged at 1500 rpm for 5 min at 23∘C and placed at 37∘C in a 5% CO2 incubator for 4 hours, after which they were centrifuged at 1500 rpm for 5 min at 23∘C. Then cells were washed with once with 1X DPBS and resuspended in complete media. Single cell suspensions were prepared and loaded onto the 10X Genomics Chromium Controller in the BSL3 facility using the Chromium NextGEM Single Cell 5' Library & Gel Bead Kit v2 to capture individual cells and barcoded gel beads within droplets for reverse transcription. The libraries were prepared according to manufacturer instructions and sequenced on an Illumina NovaSeq 6000 with a paired-end 26x91 configuration targeting a depth of 50,000 reads per cell.