Next Generation Sequencing Facilitates Single Cell Analysis of Wild Type and Monocyte-Depleted Brain Transcriptomes

Methods: Cells isolated from the whole brains of naive wild type, infected wild type, and infected CCR2-DTR mice were sort-purified on live CD45hi cells from five biological replicates and pooled. Sort-purified cells were then processed using the 10X Genomics Chromium Controller. Cell suspensions were loaded onto the Chromium Single Cell A Chip for cell lysis and barcoding. RNA from individual cells was reverse transcribed and sequencing libraries prepared using the Chromium Single Cell 3’ Library Kit v2 following the manufacturers protocol. Samples were sequenced using an Illumina NextSeq 550 with standard 10X Genomics Configuration (26 bp x 98 bp). After sequencing, raw bcl files were processed using the cellranger mkfastq command for sample demultiplexing and conversion to .fastq files, followed by cellranger count for cell barcode and UMI deconvolution as well as mapping to the respective reference genome. Processed digital gene expression matrices were imported into R studio for analysis using the Seurat package. Samples were aligned along common sources of variation and compared using canonical correlation analysis to identify unique clusters of cells within the samples. Marker genes for each sample and cluster were identified and used for generation of downstream plots within the Seurat package. All packages are maintained to be best in class and are regularly updated to their most recent release. Results: 10 distinct cell clusters were identified and we found that two populations were found at increased proportions post-infection but were substantially reduced in the monocyte-depleted animals (CCR2-DTR). We confirmed that these populations to be monocytes and monocyte-derived cells, and valicated their immunoregulatory molecules by RT-qPCR analysis. Brain CD45hi cells mRNA profiles of adult wild type (WT) and monocyte-depleted (CCR2-DTR) mice 8 days post infection of Trichinella Spiralis