title: Naive
organism: Mus musculus
description: Protocols: 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. 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. Amplified cDNA was used for 3' RNA-seq library generation, RNA-seq libraries were prepared following the manufacturer's user guide (10x Genomics).
attributes.tag: strain
attributes.value: C57BL6J
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: collection date
attributes.value: not collected
attributes.tag: sex
attributes.value: female
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: common name
attributes.value: house mouse
attributes.tag: organism part
attributes.value: lymph node
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: cell_type
attributes.value: myeloid cell
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2024-01-11