biostudies-arrayexpressSoren MullerMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13698Given the importance of sustained antigen presentation in maintenance of lymph node (LN) immune responses, we hypothesized that vaccine antigen availability and antigen-presenting cell (APC) populations may affect LN expansion. Compared to LNs of mice given the full MPS vaccine, LNs of mice given an MPS vaccine without antigen became prominently less enlarged and contracted sooner.To identify potential mediators of this differential, antigen-dependent response, we next focused the analysis of our scRNA-seq dataset on LN APC populations.biostudies-arrayexpressNucleic Acid Extraction - Sorted cells were loaded onto a 10x Chromium Chip using reagents from the Chromium Single Cell 3’ Library Kit (10x Genomics) according to the manufacturer’s protocol.Library Construction - Amplified cDNA was used for 3’ RNA-seq library generation, RNA-seq libraries were prepared following the manufacturer’s user guide (10x Genomics).Sequencing - Each single-cell RNA-seq library was sequenced on a NovaSeq S4-200 cell (Illumina).Sample Collection - Mice were treated with an MPS or bolus vaccine (containing GM-CSF, CpG, and OVA) and draining LNs were collected on day 21/20 (MPS and bolus) and compared to naïve controls. LNs were digested in RPMI-1640 (Corning) containing 0.8mg/mL Dispase II, 0.2mg/mL Collagenase P, and 0.1mg/mL DNase I (all Roche, procured from Sigma) until no visible LN pieces remained and passed through a 70μm filter. Samples were incubated in TruStain FcX (Fc block, 1:200, BioLegend) prior to incubation with staining antibodies (PE/Cy7 anti-mouse CD45, BV711 anti-mouse CD3, BV421 anti-mouse CD19, FITC anti-mouse CD31, and APC anti-mouse podoplanin, all BioLegend) and one Hashtag antibody per replicate using BioLegend TotalSeq-B03 (catalog numbers 155831, 155833, 155835, 155837, 155839; barcode sequences ACCCACCAGTAAGAC (-B0301), GGTCGAGAGCATTCA (-B0302), CTTGCCGCATGTCAT (-B0303), AAAGCATTCTTCACG (-B0304), CTTTGTCTTTGTGAG (-B0305), all diluted 1:100 in FACS buffer). Cell viability was assessed through eBioscience Fixable Viability Dye eFluor 780 (ThermoFisher). Cells were gently washed and resuspended in PBS + 2% FBS prior to sorting. Myeloid (viable, CD45+CD3-CD19-) and stromal (viable, CD45- FSC-Ahi) cells were enriched using a BD FACS Aria cell sorter into RPMI + 30% FBS on ice. Cells were counted and resuspended at an adequate concentration for loading.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Single-cell RNA-seq data for each sample were processed with CellRanger count using a custom reference package based on mouse reference genome GRCm38 and GENCODE gene models. Ambient RNA correction was performed with DecontX. In each sample cells were assigned to individual replicated using DemuxEM run on the HTO count matrix. Individual count tables were merged in R and downstream analysis was performed with the Seurat package. Counts of transcripts measured as unique molecular identifiers (UMIs) in each cell were normalized and log transformed to log(CPM/100+1) [CPM=UMI counts per million].MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsMus musculusSoren MullerfalseMPS vaccination alters long-term gene expression of antigen-presenting cells in lymph nodesGiven the importance of sustained antigen presentation in maintenance of lymph node (LN) immune responses, we hypothesized that vaccine antigen availability and antigen-presenting cell (APC) populations may affect LN expansion. Compared to LNs of mice given the full MPS vaccine, LNs of mice given an MPS vaccine without antigen became prominently less enlarged and contracted sooner.To identify potential mediators of this differential, antigen-dependent response, we next focused the analysis of our scRNA-seq dataset on LN APC populations.2024-04-05T00:00:00Z2024-04-01T17:00:19.037Z2024-01-11T22:01:14.525ZE-MTAB-13698ERP156598EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184