Developing drugs to help fight RiftValleyfevervirus Livermore_Lab PLOSPathogens
Single cell RNA sequencing
Mice were euthanized and perfused as described above. Isolated brain tissues were immediately processed using a modified protocol from []. Cortices were dissected in cold Hibernate A medium and sliced to approximately 0.5 mm before transferred to a 15 mL falcon tube with Hibernate A and B27 medium .
Cell pellets were resuspended in PBS with 0.04% non-acetylated BSA and counted on a Countess II automated cell counter prior to single-cell sequencing preparation using Chromium Single-cell 3′ GEM, Library & Gel Bead Kit v3 on a 10× Genomics Chromium Controller following manufacturers’ protocol. Sequencing data was demultiplexed, quality controlled, and analyzed using Cell Ranger and Seurat [].
]. Genes differentially expressed between clusters were identified using ‘FindMarkers’ function implemented in Seurat.Data were analyzed and graphed using Prism software. For qPCR, significance was determined using a 2-way ANOVA test comparing average expression between genotypes or gene expression changes beyond the 95% confidence intervals of infected and uninfected samples.
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