Genomics chromium single cell controller

8-week old WT and Irg1^−/− C57BL/6NJ mice were intranasally infected with 2 × 10⁷ USA300 LAC in 50 μL PBS or treated with PBS alone. 24 h after infection, mice were euthanized and their lungs were harvested. To create a single cell suspension, the lungs were placed in an Eppendorf tube containing an enzymatic digestion solution of 2 mg/mL collagenase I, 20 mg/mL dispase, 1 mg/mL elastase, and 1 μL/mL DNAse in PBS. The lungs were minced in the Eppendorf tube, then incubated with shaking at 37°C for 30 min. The digestion was quenched with 4 volumes of PBS supplemented with 10% hiFBS, and strained over a 70 μm cell strainer. The cell suspension was centrifuged at 1400 × g for 7 min at 4°C. Red blood cell lysis was performed with the eBioscience RBC lysis buffer. The resulting cell pellet was resuspended in PBS supplemented with 0.04% bovine serum albumin before being loaded onto the 10X Genomics Chromium Single Cell Controller. Cell viability was above 95% for each sample. Approximately 5000 cells were analyzed per sample. FASTQ file generation, alignment, filtering, barcode counting, and UMI counting were performed with the 10X Cell Ranger software.