title: GSM9527708: Lungs from mice with a low metastatic burden; Mus musculus; RNA-Seq
library_construction_protocol: Mice were perfused with 10mL 1X Hank's balanced salt solution (HBSS; Corning CellGro, No. 21-023-CV). Lungs were collected and rinsed in chilled 1X PBS. The tissue was cut into small pieces with a razor blade and incubated in 5mg/mL type 2 collagenase (Worthington Biochemical Corporation, No. LS004174) in phenol-red free DMEM for 10min at 37ºC on a benchtop shaker. Enzyme A/Buffer Y mix from the Neural Tissue Dissociation Kit P from Miltenyi Biotec (No. 130-092-628) was added to the collagenase-tissue mixture and incubated for another 15min at 37ºC on a benchtop shaker. Pipetting periodically during this incubation was used to dissociate the tissue. Ice cold PBS/bovine serum albumin (BSA) cell buffer (1X PBS with 0.5% 50mg/mL BSA; ThermoFisher Scientific, No. AM26160) was used to halt the dissociation enzymatic reaction. Cells were passed through a 100μm mesh filter and centrifuged at 300 xg for 5min at 4ºC. Cells were then resuspended in ammonium-chloride-potassium (ACK) buffer for 1min at 20ºC to lyse any remaining red blood cells. Cold PBS/BSA cell buffer was added and cells centrifuged at 300 xg for 10min at 4ºC. Cells were resuspended in cold 1X PBS and debris was removed using cold Debris Removal Solution (Miltenyi Biotech, No. 130-109-398) according to the manufacturer's instructions. Cells were washed again with PBS/BSA cell buffer and centrifuged at 300 xg for 5min at 4ºC. Finally, cells were resuspended in 500μL-1mL PBS/BSA cell buffer and filtered through a 40μm Flowmi cell strainer (Millipore Sigma, No. BAH136800040). Approximately 10,000 cells were used to generate libraries and sequenced by the Genomics Shared Resource (RRID:SCR_021984) at the University of Colorado Anschutz Medical Campus.