20.500.12556/RUL-181084 Deep single-cell transcriptomic profiling of bovine milk somatic cells revealed expression of stem cell related transcription factors Background/Objectives: Milk somatic cells reflect the cellular composition and functional state of the lactating mammary gland and represent a valuable, non-invasive source for transcriptomic studies. Single-cell RNA sequencing (scRNA-seq) enables cell-type-resolved analysis of bovine milk; however, sequencing depth strongly influences the detection of lowly expressed genes and the resolution of transcriptional cell states. The aim of this study was to further characterise the single-cell transcriptome of bovine milk somatic cells, with particular emphasis on high-resolution gene expression profiling and cellular heterogeneity. Methods: Milk somatic cells were isolated from two healthy Holstein Friesian cows in mid-lactation and profiled using a droplet-based scRNA-seq platform. Newly generated high-depth datasets were integrated with two previously published bovine milk scRNA-seq datasets using an identical bioinformatics pipeline. Data integration, clustering and cell-type annotation were performed using the Seurat framework, and transcription factor expression was evaluated across datasets with different sequencing depths. Results: Single-cell transcriptomic analysis revealed a diverse cellular landscape in bovine milk, comprising epithelial, progenitor, and immune cell populations. Unsupervised clustering identified 21 transcriptionally distinct clusters, including multiple CD8+ T-cell subpopulations, monocytes, neutrophils, mast cells, and B cells, as well as luminal epithelial and luminal progenitor cells. While overall cell-type composition was comparable across datasets, deeply sequenced samples exhibited higher transcriptomic complexity and enabled refined resolution of immune and epithelial subpopulations. Deeper sequencing facilitated the detection of low-abundance transcription factors that were not observed in lower-depth datasets. Among these, NANOG was detected exclusively in deeply sequenced samples, suggesting the presence of rare transcriptional states associated with cellular plasticity. Conclusions: This study expands the single-cell transcriptomic landscape of bovine milk somatic cells and demonstrates the importance of sequencing depth for resolving functional cellular heterogeneity. The results highlight milk as a powerful, non-invasive source for investigating mammary gland biology and cellular plasticity during lactation. single-cell RNA sequencing bovine milk milk somatic cells mammary gland lactation transcriptome cattleHolstein Friesian cows govedo mleko mlečna žleza somatske celice genetika matične celice transkriptomika true false true Angleški jezik Slovenski jezik Članek v reviji 2026-03-24 14:55:01 2026-03-24 14:55:11 2026-03-25 04:33:14 0000-00-00 00:00:00 2026 0 0 13 str. issue 4, [article no.] 365 Vol. 17 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2026-03-24 575:636.257 2073-4425 10.3390/genes17040365 272865539 RAZ_Dolinar_Mateja_2026.pdf RAZ_Dolinar_Mateja_2026.pdf 1 187919B34820E8C89AB665E32D60CC38 29819061fb0ce995e0a8c1efffbb59c0d6e1b5a596694cf10344d0f7bbb2cfcf 00c5a5e9-2789-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=231241 https://www.mdpi.com/2073-4425/17/4/365 1 f5dad849-2788-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=231240 Biotehniška fakulteta 0 0 0