title: GSM8213614: Flu_metastasis; Mus musculus; RNA-Seq
library_construction_protocol: Whole lungs were harvested and digested using lung digestion mix (1.5 mg/mL collagenase A (Sigma Aldrich cat# COLLA-RO; St. Louis, MO), 0.4 mg/mL deoxyribonuclease I (Worthington cat# LS002139; Lakewood, NJ), 10 mM HEPES pH 7.2, 5% FBS) was injected into the lungs through cannulae and were incubated at 37°C for 30 min. Digested lungs were passed through a 50 mm cell strainer and red blood cells were lysed using hemolytic buffer (150 mM NH4Cl, 1 mM NaHCO3, 1.1 mM Na2EDTA).Live cells were enriched using the Miltenyi dead cell removal kit (130-090-101). Cells were provided to the Genomics Shared Resource of the University of Colorado Cancer Center (RRID: SCR_021984) and subjected to library construction as follows. Cells exhibiting >80% viability are fixed in a 4% formaldehyde fixative solution and using Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10x Genomics). The whole transcriptome probe pairs (10X Genomics) are added to the fixed single cell suspensions to hybridize to their complementary target RNA in an overnight incubation at 42°C. After hybridization, the unbound probes are washed off. The fixed and probe-hybridized single cell suspensions are loaded onto Chromium X (10X Genomics) microfluidics instrument to generate partitioned nanoliter-scale droplets in oil emulsion. Each droplet contains a barcoded gel bead, a single cell, and enzyme Master Mix (10X Genomics) for probe pair ligation and gel bead primer barcode extension. The droplets in oil emulsion are place in a thermal cycler for 60 min at 25°C, 45 min at 60°C, and 20 min at 80°C. The single cell-barcoded, ligated probe product undergo library preparation to be compatible with Illumina next-generation sequencing.