title: GSM8616725: DSP-1001660015236-G-F02; Mus musculus; OTHER
library_construction_protocol: The popliteal lymph nodes were harvested either 21 or 42 days after immunization with 20mg of one or both ova-psDNA GeoMx tags and 1E4 pfu per foot of vaccinia virus. Lymph nodes were fixed for 24-hours in 10% phosphate buffered formalin, and then placed in 70% Ethanol until embedding. The lymph nodes were embedded in paraffin wax, 7 uM sections were cut using a microtome. Slides were stained with Lyve-1-647 (clone: EPR21771), CD11c-Biotin (clone: D1V9Y), CD4 (clone: 4SM95). The nuclei were counterstained with SYTO™ 82 Orange Fluorescent Nucleic Acid Stain (S11363). Biotinylated antibodies were visualized with Streptavadin-AF594 (S32356) and the CD4 antibody was visualized with OPAL 520 (FP1487001KT, Akoya Biosciences). The ROI's selected to undergo GeoMx DSP spatial transcriptomics were chosen based on Lyve-1 staining, CD11c staining and CD4 staining. Regions were selected based on the size of the area or the maximum size allowed for the R01. Sequencing libraries were generated following the Nanostring GeoMx NGS protocol.