GSM6040511: Infected d100, replicate 1, scRNAseq; Mus musculus; RNA-Seq
GSM6040511
Mice were euthanized with CO2 and lungs were perfused with heparin in saline. Lungs were minced and incubated in Collagenase/DNAse for 30 minutes at 37°C. Lung tissue was pushed through a 70 µm nylon screen to obtain a single-cell suspension. Red blood cells were lysed and the cells were resuspended in suitable media or buffer for further use. Isolated total lung single-cell suspensions were enriched for live cells using dead cell depletion kit according to manufacturer's instruction (Miltenyi Biotec) and subjected to droplet-based massively parallel single-cell RNA sequencing using Chromium Single Cell 5' (v1) Reagent Kit as per manufacturer's instructions (10x Genomics). Briefly, cell suspensions were loaded at 1,000 cells/µL with the aim to capture 10,000 cells/lane. The 10x Chromium Controller generated GEM droplets, where each cell was labeled with a specific barcode, and each transcript labeled with a unique molecular identifier (UMI) during reverse transcription. The barcoded cDNA underwent 16 cycles of amplification, and after SPRI bead purification, was removed from the BSL-3 space for library generation. Libraries were prepared from amplified cDNA and target enrichment products by fragmentation, end repair, A-tailing, adapter ligation, and sample index PCR as per the manufacturer's instructions. Libraries were sequenced on a NovaSeq S4 (200 cycle) flow cell, targeting 50,000 read pairs/cell.