title: GSM9161817: HMPV, nasal turbinates replicate 2, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Cells were collected from mice euthanized day 1 post-infection. Following collection, nasal turbinates were flushed twice with 1-mL R10 (RPMI 1640 media supplemented with 10% FBS, 2 mM glutamine, 50 μg/ml gentamicin, 2.5 μg/ml amphotericin B, and 50 μM β-mercaptoethanol). Nasal turbinates were minced and digested with 2 mg/mL collagenase, 20 μg/mL DNase (Roche), and 2 U/mL dispase (Corning) for 60 minutes in a shaker at 37C. Nasal turbinate preparations were passed through a 70-μM cell strainer and incubated with ACK lysis buffer (Sigma-Aldrich) to remove red blood cells. Cells were washed 5 times with 10mL of PBS+10% FBS and centrifuged at 500xg for 5', followed by a final wash and centrifugation at 200xg for 10'. Dead cells were removed by magnetic separation using Annexin V kit (STEMCELL Technologies). The 10X Genomics 3' CellPlex Kit was used and cells were tagged by lipid-conjugated Barcode oligonucleotides according to manufacturer's instructions. Cells were passed through a 40-μM filter and cell viability was determined using Cellometer 2000 (all showing >90% viability) prior to loading on 10X Chromium instrument. Following established techniques using Chromium Single Cell 3' Library V2 kit (10X), libraries were constructed and RNA-seq was performed on each sample targeting 20,000 reads per cell.