title: Tppp3 KO
organism: Mus musculus
description: Protocols: Tracheae were dissected from 10-week-old wild-type and Tppp3 knockout mice. Tissues were incubated in Dispase (16 U/ml) at 37 °C for 40 min to detach the epithelium. The epithelial sheets were peeled off and incubated in 0.25% Trypsin–EDTA at 37 °C for 60 min. After incubation, the suspension was passed through a 40 µm cell strainer to remove residual tissue and centrifuged at 400 × g for 5 min. The pellet was resuspended in 1× RBC lysis buffer and left at room temperature for 3 min, then centrifuged again at 400 × g for 5 min. The resulting cell pellet was collected for downstream single-cell RNA-seq library preparation. Not applicable. Total RNA was not extracted. Single cells were loaded onto the 10x Genomics Chromium Single Cell 3′ v3 workflow, where cells were lysed in GEMs and poly(A) mRNA was captured with oligo-dT barcoded beads. Reverse transcription and cDNA cleanup were performed according to the manufacturer's instructions; therefore no bulk nucleic acid extraction was carried out prior to library construction. Single-cell RNA-seq libraries were constructed using the 10x Genomics Chromium Single Cell 3′ v3 Reagent Kits according to the manufacturer's protocol. Briefly, single cells were encapsulated into Gel Beads-in-Emulsion (GEMs) using the Chromium Controller, and mRNAs were reverse-transcribed to generate barcoded cDNAs containing unique molecular identifiers (UMIs) and cell barcodes. After reverse transcription, GEMs were broken, and cDNA was purified and amplified by PCR. Amplified cDNA was fragmented, end-repaired, A-tailed, and ligated with Illumina sequencing adapters to generate the final sequencing library. Library quality and concentration were verified using an Agilent Bioanalyzer and Qubit fluorometer before sequencing.