biostudies-arrayexpressLijiang FeiHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16728Plasmacytoid dendritic cells (pDCs) are a rare immune cell population that plays a key role in antiviral innate immunity. In our analysis, secretin (SCT) emerged as one of the most highly expressed hormones in both circulating and tissue-resident pDCs. To validate this finding, we performed spatial transcriptomic analysis of adult tonsil tissue, which demonstrated co-localization of SCT transcripts with the pDC marker LILR4A. We further confirmed SCT expression at the protein level by immunofluorescence staining of primary human peripheral blood pDCs, showing co-expression of secretin with the canonical pDC markers CLEC4C and TLR9. In a longitudinal single-cell RNA-sequencing dataset from individuals with COVID-19, we observed modest upregulation of SCT in interferon-activated pDCs. To determine whether SCT expression is linked to immune function, we purchased primary human peripheral blood pDCs and stimulated them in vitro with the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide, followed by single-cell transcriptomic profiling.biostudies-arrayexpressLibrary Construction - Single-cell suspensions were prepared in advance and kept at 4 °C or on ice throughout barcoding. To enable multiplexing, cells were labeled with unique DNA barcodes using Anchor and Co-Anchor oligonucleotides (5 μM each in 1% BSA/PBS). Up to 0.5 million cells per sample were processed. Cells were washed in 2% BSA/PBS, pelleted, and resuspended in 1% BSA/PBS. Each sample was incubated sequentially on ice with 10× Anchor:Barcode solution (20 μL) followed by 10× Co-Anchor solution (20 μL), with gentle pipette mixing and 5-minute incubations between steps. After labeling, cells were washed once in 1% BSA/PBS, pelleted, and pooled in equal cell numbers. Pooling was performed by serially combining samples with 350 μL of PBS/BSA per transfer to maximize recovery. The pooled suspension was centrifuged, and cells were resuspended in a minimal volume of buffer for counting and loading into the 10x Genomics Chromium platform for 3′ gene expression profiling. The pooled, barcoded single-cell suspension was then processed using the 10x Genomics Chromium platform following the manufacturer’s protocol for 3′ gene expression library preparation via Single Cell 3' v4 (polyA).Nucleic Acid Extraction - Primary human plasmacytoid dendritic cells (pDCs) were isolated from peripheral blood (PB) mononuclear cells (MNCs) using negative immunomagnetic separation techniques. PB was collected using acid-citrate-dextrose solution A (ACDA) as the anticoagulant.Sequencing - Libraries were sequenced on NovaSeq X.Growth Protocol - Isolated pDCs were cultured in ImmunoCult™-XF Medium (STEMCELL Technologies, #10981). Cells were maintained at 37 °C in a humidified incubator with 5% CO₂.Sample Collection - Plasmacytoid dendritic cells (pDCs) were purchased as pre-isolated cells (STEMCELL Technologies, #70046).Sample Treatment - Stimulation with CpG oligonucleotides (InvivoGen, #tlrl-kit9h) was performed on day 1 post isolation at a final working concentration of 6 μg/mL for 24 hours prior to downstream analyses.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Sequencing reads were aligned to the human reference genome (GRCh38-2020-A-2.0.0) using Cell Ranger (v9.0.1). Ambient RNA contamination was removed using CellBender (v0.3.0). For lanes containing pooled genotypes from multiple donors, cell barcodes were demultiplexed by genotype using BAM files as input to souporcell (v2.5). Two sequencing libraries were processed, each containing four samples derived from four distinct donors. The donors had unique demographic characteristics: one female, one of European ancestry, and one with blood type O−. Using the souporcell results, alternative allele fractions were calculated at each variant site as ALT / (REF + ALT), and a cell-by-variant matrix was constructed. Variant annotations were obtained from common_variants_covered.vcf, and souporcell donor assignments were included as cell-level metadata. Cells were clustered based on allele fraction profiles across informative variants. Cells originating from the same donor clustered together, enabling cross-library donor matching. To infer ancestry, we identified the top differential variants for each donor and selected sites with |EUR_AF − AMR_AF| > 0.25 as ancestry-informative markers, where EUR_AF and AMR_AF represent allele frequencies in European and Admixed American populations, respectively. Using these variants, we applied sc.tl.rank_genes_groups to score each cell. One donor showed strong enrichment for European-informative variants and was therefore assigned to European ancestry. Sex and blood type were determined using expression-based markers. Clustering of the single-cell expression matrix identified one donor with high XIST expression, consistent with female sex. Because Rh-negative individuals typically carry a complete deletion of RHD, and only one donor lacked detectable RHD expression, that donor was assigned as O−. Then, using scanpy (1.9.3), cells were filtered to retain those with >200 detected genes, >500 UMIs, mitochondrial gene content <20%, and a doublet score <0.3 as inferred by Scrublet. Filtered cells were log-normalized and clustered using the Leiden algorithm (resolution 0.2). Cell types were annotated using CellTypist and manually validated based on canonical marker genes. Only singlet cells assigned by souporcell were retained for downstream analyses.Sequence Alignment - Sequencing reads were aligned to the human reference genome (GRCh38-2020-A-2.0.0) using Cell Ranger (v9.0.1).UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XnoneRNA-seq of coding RNA from single cellsHomo sapiensLijiang FeiSarah TeichmannfalseSingle-cell RNA-seq of human primary pDCs before and after 24 h stimulation with TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotidePlasmacytoid dendritic cells (pDCs) are a rare immune cell population that plays a key role in antiviral innate immunity. In our analysis, secretin (SCT) emerged as one of the most highly expressed hormones in both circulating and tissue-resident pDCs. To validate this finding, we performed spatial transcriptomic analysis of adult tonsil tissue, which demonstrated co-localization of SCT transcripts with the pDC marker LILR4A. We further confirmed SCT expression at the protein level by immunofluorescence staining of primary human peripheral blood pDCs, showing co-expression of secretin with the canonical pDC markers CLEC4C and TLR9. In a longitudinal single-cell RNA-sequencing dataset from individuals with COVID-19, we observed modest upregulation of SCT in interferon-activated pDCs. To determine whether SCT expression is linked to immune function, we purchased primary human peripheral blood pDCs and stimulated them in vitro with the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide, followed by single-cell transcriptomic profiling.2026-05-22T00:00:00Z2026-05-22T13:00:01.518Z2026-03-05T13:08:54.681ZE-MTAB-16728ERP189952EFO_0002944EFO_0004170EFO_0003789EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969