title: GSM8842519: RSV-corn oil rep2; Mus musculus; Human respiratory syncytial virus A strain Long; RNA-Seq
library_construction_protocol: The right lung was digested in Dispase II (100mg/mL), Collagenase A (10mg/mL), DNAse I (1500 kU/mL), and CaCl2 (0.25M) into a single cell solution using a 40 mm filter to create a single cell solution. Half the cells were separated for Single Cell Sequencing. Libraries were constructed according to the Chromium NextGEM Fixed RNA Profiling Reagent Kits user guide (10x Genomics). Cells frozen in 10X Genomics Quenching Buffer were thawed and resuspended in 0.5X PBS with 0.02% BSA. The total cell number was quantified on the Luna-FX7 automated cell counter (Logos Biosystems, Anyang, South Korea) using acridine orange/propidium iodide stain. Aliquots of less than two million cells were used as input for the probe hybridization reaction and incubated at 42C overnight (21 hours 16 minutes). Following overnight incubation, subsets of four samples with unique probe barcodes were pooled together and washed three times with 10X Genomics Post-Hyb Wash Buffer. Samples were filtered through 30 μm Celltrics filters (Sysmex, Kobe, Japan) and the appropriate volume of cells was loaded onto a Single Cell Chip Q required for recovery of [enter captured cell number] cells.  Following the completion of the Chromium run the GEMs were transferred to emulsion safe strip tubes for the cell and UMI barcoding reaction using an Eppendorf MasterCycler Pro thermocycler (Eppendorf, Hamburg, Germany). After the GEM incubation, GEMs were broken, and the barcoded probe products were amplified by PCR. A portion of the amplified probe products was put into a second PCR reaction to add unique dual indexes to each library. The final libraries were quantified on the Qubit v4 fluorometer (ThermoFisher Scientific) and profiled on the Agilent 4200 Tapestation (Agilent Technologies, Santa Clara, CA). These libraries were sequenced on a NovaSeq X+ sequencer with paired-end, 150bp sequencing. Data was processed with bcl2fastq.