biostudies-arrayexpressXiangning DongHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14953We performed micro-dissections of adult human lung obtained through Gift of Life and performed single nuclear RNA-sequencing (n = 2 biological replicates, age 20, 26 yo) on trachea, bronchi, and bronchioles.biostudies-arrayexpressSample Collection - Human lung tissue research was reviewed and approved by The University of Michigan Institutional Review Board (IRB). Normal, de-identified human fetal lung tissue was obtained from the University of Washington Laboratory of Developmental Biology. Tissue was shipped overnight in UW-Belzer’s solution (Thermo Fisher, Cat#NC0952695) on ice and was processed for experiments or fixation within 24h. Normal, de-identified, donor human adult lungs not eligible for transplant were obtained from Gift of Life Michigan Donor Care Center. Specific tissue regions were dissected according to anatomic region and snap-frozen in liquid nitrogen for further downstream analysis.Nucleic Acid Extraction - Preparation was performed as described previously in accordance with 10X Genomics’ protocols CG000505 Rev A for adult tissue). Briefly, snap-frozen tissue was minced into smaller fragments using a scalpel and then added to lysis buffer (Thermo Fisher Scientific, Cat#PI28324). Tissue was homogenized using a pellet pestle 15 times then incubated for 5 minutes in lysis buffer. The cell suspension was filtered through a 33 μm strainer. Suspension was centrifuged at 500g for 5 mins at 4°C. Supernatant was removed and the pellet was washed with PBS + 1% BSA, centrifuged at 500g for 5 mins at 4°C; pellet was washed and centrifuged again. An added debris removal column step was used for the adult tissue. Permeabilization for ATAC-seq samples was performed by incubating the pellet in 0.1X lysis buffer and incubated for 2 minutes. The suspension was then centrifuged at 500g for 5 mins at 4°C; supernatant was removed and pellet was resuspended in diluted nuclei buffer.Library Construction - Immediately after tissue dissociation, single nucleus libraries were prepared on the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 16 rxns PN-1000283 was used with a target of 7500 cells.Sequencing - All single-nucleus RNA-sequencing was performed using Illumina Novaseq 6000 by the University of Michigan Advanced Genomics Sequencing Core.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - All gene expression datasets underwent independent normalization, variable feature selection, scaling and principal component analysis (PCA). Anchors for batch-corrected integration were determined using a reciprocal PCA approach considering 30 principle components (PCs) from each dataset. Integrated data matrices were produced, scaled, subjected to PCA, which formed the basis for UMAP projection (30 PCs), cell-neighbor identification (30 PCs) and cell clustering (resolution = 0.75). At this stage a cluster of cells in the mesenchyme defined by higher mitochondrial and ribosomal content than all other clusters was removed, as well as a cluster of cells in the Epithelium that was contaminated with mesenchymal marker expression. After removal of these low-confidence cell clusters data was freshly integrated by reciprocal PCA, dimensionally reduced and clustered as above.UnknownTranscriptomicsGenomicsProteomicsN/A10X Genomics reagentsIllumina NovaSeq 6000Chromium Next GEM Single Cell Multiome Reagent Bundlesingle nucleus RNA sequencingHomo sapiensXiangning DongYusoo LeeTristan FrumPeggy HsuJason SpencefalseSingle-nucleus RNA sequencing of healthy human adult lung from trachea, bronchi and bronchiolesWe performed micro-dissections of adult human lung obtained through Gift of Life and performed single nuclear RNA-sequencing (n = 2 biological replicates, age 20, 26 yo) on trachea, bronchi, and bronchioles.2025-08-01T00:00:00Z2025-03-19T17:26:57.229Z2025-03-19T17:26:57.229ZE-MTAB-14953ERP170561EFO_0002944EFO_0004170EFO_0009809EFO_0005518EFO_0003816EFO_0004184