title: GSM8033307: WT_trachea; Mus musculus; RNA-Seq
library_construction_protocol: 5-8 month old mice were humanely euthanized and the tracheas removed. They were digested using Pronase solution (Roche) and plated on Primaria cell culture dishes for three to four hours to separate the epithelial cells from the fibroblasts. The Miltenyi Biotec Dead Cell Cleanup Kit was used to remove dead cells before passing through a 40 um filter and collection of live cells using a MiniMACS Separator column (Miltenyi Biotec). Cells were resuspended in Hams F-12 media with 10% fetal bovine serum to a final count of 1x10^6 cells/ml. 8500 cells were loaded into Chromium Next GEM Chip G (10X Genomics, #1000120) in the reverse transcription reaction according to the manufacturer's instructions to capture at least 5000 cells.  Reverse transcription, gel beads-in-emulsion (GEM) generation, and library preparation were performed using the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics, #1000268). Gel beads were loaded into the designated wells of the same Chip G, which was then inserted into the Chromium Controller for GEM generation.  Following GEM generation, full-length barcoded cDNAs were produced from poly-adenylated mRNA by reverse transcription, and the cDNA was amplified using twelve amplification cycles per manufacturer recommendation.  The amplified cDNA was cleaned up using SPRIselect (Beckman Coulter), and 1 µl of the cDNA was run on a bioanalyzer using the High Sensitivity DNA kit (Agilent, #5067-4627) to assess the quality and determine concentration.  To prepare Illumina sequencing-compatible cDNA libraries, the cDNA was subjected to fragmentation, followed by end repair, A-tailing, and adapter ligation.  After each of these steps, the reaction product was cleaned up using SPRIselect.  The samples were then amplified with dual sample indexes to enable multiplexing during sequencing, cleaned up, and quantified.  The cDNA was adjusted to a concentration of 0.5 ng/ml and sequenced.