GSM8116314
One lobe of the lungs was minced and subjected to Liberase TL digestion (0.25 mg/mL) in the presence of DNaseI (0.5 mg/mL) at 37°C for 1 hour. Cells from 2 mice per group were pooled, treated with ACK lysing buffer for 1 minute, washed, passed through a 70-µm cell strainer, and counted using the TC20 Automated Cell Counter (BioRad). Subsequently, cells were processed on the Rhapsody platform following the manufacturer's protocol. Cells from each treatment group were labeled with individual sample tags (BD® Mouse Single-Cell Multiplexing Kit (Cat. No. 633793)). Before loading into the cartridges, cells were quantified on the Rhapsody scanner following staining with Vybrant® DyeCycle™ Green (Invitrogen, V35004) for 5 minutes at room temperature. Barcoded samples were pooled in equal amounts for each of the four treatment groups (Naïve, LNP, mRNA, mRNA + Ev.), and sixty thousand cells were loaded into each cartridge, with two cartridges used for sequencing. Single cells were isolated with the BD Rhapsody Express Single-Cell Analysis System according to the manufacturer's recommendations; after the final bead wash step, forty thousand cells were detected as singlets. Libraries were prepared following the BD Rhapsody System mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol (BD Biosciences; 633801) and sequenced at the CCHMC Genomics Sequencing Facility on NovaSeq 6000 using PE (paired end) 100 sequencing format. Following sequencing and preprocessing by the Rhapsody WTA analysis pipeline using exact cell count, 28,000 cells per cartridge were used for the downstream analysis.