title: GSM9504717: dorsal trachea, control group; Mus musculus; RNA-Seq
library_construction_protocol: Tracheal cells were dissociated using a papain-based enzymatic digestion protocol. For whole-trachea cell isolation, each trachea was longitudinally bisected and further cut into pieces, then incubated in papain dissociation solution (Worthington) prepared in EBSS (5 mL per three tracheae) supplemented with DNase I. Samples were incubated at 37 °C for 45 minutes. Following digestion, tissues were mechanically triturated to release single cells, filtered through a 100-µm cell strainer, and centrifuged at 500 × g for 5 minutes at 4 °C. Cell pellets were resuspended in EBSS containing ovo-mucoid protease inhibitor and DNase I and incubated on a rotating platform at 4 °C for 20 minutes to quench residual papain activity. Cells were then subjected to red blood cell lysis, washed, collected by centrifugation. scRNA-seq libraries were generated according to the manufacturer's instructions using the Chromium GEM-X Single Cell 5′ v3 Gene Expression platform (10x Genomics). Briefly, single-cell suspensions were resuspended in the master mix and loaded together with gel beads and partitioning oil into a Chromium chip to generate Gel Beads-in-Emulsion (GEMs).Polyadenylated RNA transcripts from individual cells were reverse-transcribed into cDNA incorporating Illumina read 1 sequences, a cell barcode, and a unique molecular identifier (UMI). After GEM recovery, cDNA was amplified by PCR, and amplified products were purified using Silane Dynabeads. Size selection was performed using SPRIselect reagent to obtain appropriately sized fragments for library construction. During library preparation, Illumina R2 sequences, P5 and P7 adapters, and sample indices were added.