GSM8427131: 2RTB0113, LTBI, MtbNEG, 24h, BAL, scRNA-seq; Homo sapiens; RNA-Seq
Bronchoalveolar lavage (BAL) were collected and cryopreserved in 10% dimethyl sulfoxide (MilliporeSigma, Massachusetts, USA) and 90% fetal bovine serum (Cytiva, Massachusetts, USA) and, by gradual cooling to − 80 °C in a Nalgene Mr Frosty™ container [Sigma Aldrich (Pty) Ltd, Gauteng, South Africa] with isopropanol for 24 h followed by long term preservation in liquid nitrogen. Cryopreserved BAL samples were set in culture following dropwise thawing protocol. Then, BAL cells were cultured with media (not challenged, MtbNEG) or media + Mtb strain H37Rv at MOI 6.5:1 (challenged, MtbINF) for 6 or 24h at 37°C, 5% CO2, and 95% relative humidity. Single cell capture and library preparation was performed with Chromium Next GEM Single Cell 3ʹ Reagents Kit v3.1 (10X Genomics, USA). Cell suspensions were loaded on a Chromium Next GEM Chip G (10X Genomics, USA) together with gel bead from Chromium Next GEM Single Cell 3ʹ GEM Kit v3.1 and captured on Chromium Controller (10X Genomics, USA) with recovery target of 1×104 cells. One quarter of the total cDNA was used to generate sequencing libraries using Library Construction Kit (10X Genomics, USA) and barcoded using Dual Index plate TT set A (10X Genomics, USA). Obtained libraries were double sided size selected using SPRIselect beads (Beckman Coulter, USA) to enrich for fragments 300-800 base pairs long, centered at 450bp. Libraries were checked for quality with Bioanalyzer High Sensitivity DNA Kit (Agilent, USA).