GSM8150607: pd-1 ko lungs, M.tb-infected, 7days; Mus musculus; RNA-Seq
Lungs were aseptically removed and homogenized in PBS containing DNase Ⅰ (EN401; Vazyme) and type Ⅰ collagenase (17100017; ThermoFisher), and incubated at 37℃ for 60 min. The homogenates were filtered through a 70μm cell strainer and centrifuged at 1500 rpm for 5 min. Red blood cells were lysed using red blood lysing buffer, and cells were pelleted and resuspended in RPMI-1640 medium for library preparation. The single-cell suspension derived from dissociated lung tissues was analyzed for viability, and loaded onto the 10X Genomics Chromium Single Cell Controller at a concentration of one thousand cells per microliter. Next, the cell suspension, reagents, and 10x Genomics gel beads were encapsulated into individual nanoliter-sized Gelbeads in Emulsion (GEMs) and then reverse transcription of poly-adenylated mRNA was performed inside each droplet. The cDNA and VDJ-enriched libraries were completed in a single bulk reaction using the 10X Genomics Chromium NextGEM Single Cell 50 v 1.1 and V(D)J Reagent Kits. 50,000 or 5,000 sequencing reads per cell for scRNA-seq or VDJ libraries, respectively, were generated on the Illumina NextSeq 500 instrument.