biostudies-arrayexpressTakafumi SakaiMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16187Single-cell RNA-seq was performed on tracheal epithelial cells from wild-type and Tppp3 knockout mice using the 10x Genomics Chromium Single Cell 3′ v3 platform. Seven major epithelial cell types were identified, and no novel cell type was detected in Tppp3 KO trachea. Expression of cilia-related genes (Mcidas, Foxj1, Myb, Ccno, Rfx2, Rfx3) showed no significant changes, suggesting that differentiation of tracheal ciliated cells was not affected by Tppp3 loss.biostudies-arrayexpressLibrary 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.Sample Collection - 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.Sequencing - Libraries were sequenced on an Illumina NovaSeq 6000 platform with paired-end reads (Read 1: 28 bp, i7 Index: 8 bp, Read 2: 91 bp) according to the 10x Genomics Chromium Single Cell 3′ v3 recommendations. Read 1 contained the 16-bp cell barcode and 12-bp UMI sequence, and Read 2 corresponded to the cDNA fragment derived from poly(A) mRNA. Sequencing data were demultiplexed and converted to FASTQ files using Illumina bcl2fastq software.Nucleic Acid Extraction - 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.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Processed data were generated using Cell Ranger (10x Genomics, version 6.0). Raw FASTQ files were aligned to the mm10 reference genome using STAR within the Cell Ranger pipeline. UMI counts per gene per cell were calculated to produce a gene-by-cell expression matrix. Only barcodes passing Cell Ranger’s quality control thresholds were retained in the filtered matrix.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsMus musculusTakafumi SakaiKyosuke ShinoharafalseSingle-cell RNA-seq analysis of mouse tracheal epithelial cells from wild-type and Tppp3 knockout miceSingle-cell RNA-seq was performed on tracheal epithelial cells from wild-type and Tppp3 knockout mice using the 10x Genomics Chromium Single Cell 3′ v3 platform. Seven major epithelial cell types were identified, and no novel cell type was detected in Tppp3 KO trachea. Expression of cilia-related genes (Mcidas, Foxj1, Myb, Ccno, Rfx2, Rfx3) showed no significant changes, suggesting that differentiation of tracheal ciliated cells was not affected by Tppp3 loss.2025-12-30T00:00:00Z2026-05-27T14:34:25.264Z2025-11-18T23:53:58.513ZE-MTAB-16187ERP185272EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184