GSM8231019: bio replicate 2, gene expression [L6b]; Mus musculus; RNA-Seq
TCR⍺ KO mice were injected intravenously (IV) with ESAT-6 specific (C7 and C24) TCR transgenic T-cells and infected with Mtb Erdman 24 hours post-IV. At 26 wpi, mice were IV injected with anti-CD45 antibody 2 minutes before euthanizing with CO2. Lungs were then perfused and removed 3 minutes after anti-CD45 injection. Lung single cell suspensions were prepared by homogenizing lungs using a GentleMACS tissue dissociator, digesting with collagenase at 37C for 30 minutes, followed by a second run of dissociation using the GentleMACS. Suspensions were then filtered through 70 µm strainers and incubated with 1 mL ACK Lysis Buffer (GIBCO) for 1 minute at room temperature (25C). The cells were resuspended in D-PBS and filtered through 40 µm filters. The resulting single cell suspensions were stained with flow antibodies to sort for total live, parenchymal T-cells (CD45 IV Negative, TCR[b] and CD3 double positive) using the BD FACS Aria. Library construction was performed according to the manufacturer's instructions (single cell 5' v2 protocols, 10x Genomics).