GSM8993134: Pooled sample tags 1-12; Mus musculus; RNA-Seq
Mice were culled at the indicated day post-infection (dpi) in each experiment, including dpi 0 (mock), 3, 5 and 7.  The brain and bone marrow (BM) were collected from mice anesthetized with an intraperitoneal injection of avertin and perfused transcardially with PBS. Cells from the BM were isolated by flushing the femur with PBS using a 30-gauge needle. Red blood cells were then lysed using 1x Pharm Lyse Buffer (BD Biosciences, USA). Brains were digested enzymatically with DNase I (0.1 mg/mL, DN25, Sigma-Aldrich, USA) and collagenase type IV (1 mg/mL, C5138, Sigma-Aldrich, USA) using the gentleMACS dissociator (Miltenyi Biotec, DE). A 30/80% Percoll gradient was then used to isolate leukocytes from brain homogenates. Live cells were counted with trypan blue (0.4%) on a hemocytometer. Monocytes were isolated from the BM and microglia and monocyte-derived cells (MCs) were isolated from the brain at the designated dpi. To ensure ample cells for sorting, tissues from two animals were combined per sample. Single cell suspensions underwent CD16/32 blocking and Zombie UV viability staining (Biolegend, USA) before incubation with a cocktail of fluorescently-conjugated surface stain antibodies. Sorting was performed on a 10-laser Influx Cell Sorter (BD Bioscences, USA) using the FACS Diva Program (BD Biosciences, USA). In this experiment, microglia and MCs were isolated from the brain; microglia were defined and sorted as Ly6G⁻, CD49dlo, P2RY12hi, CD11b⁺, and CX3CR1⁺, while MCs were Ly6G⁻, CD49dhi, P2RY12lo, NK1.1⁻, CD3e⁻, CD11b⁺, CD64⁺, and CX3CR1⁺. Mature monocytes from the BM at 7 dpi were identified as CD45.2+, Ly6G-, CD48hi, NK1.1-, CD3e-, B220-, CD11b+, CD117-, CD115hi. Sorted samples were then individually barcoded with sample tagd using a mouse multiplexing kit using anti-CD45 (BD Biosciences, USA). Sample tags are labelled as follows: #1 Brain microglia, dpi 0 (mock) rep1; #2 Brain microglia, dpi 0  (mock)rep2; #3 Brain microglia, dpi5 (WNV) rep1; #4 Brain microglia, dpi (WNV) 5 rep2; #5 Brain microglia, dpi 7 (WNV) rep1; #6 - Brain microglia, dpi 7 (WNV) rep2; #7 Brain MCs, dpi 5 (WNV) rep 1; #8 Brain MCs, dpi 5 (WNV) rep2; #9 Brain MCs, dpi 7 (WNV) rep1; #10 Brain MCs, dpi 7 (WNV) rep2; #11 - BM monocytes, dpi 7 (WNV) rep 1; #12 BM monocytes, dpi 7 (WNV) rep2. Cells were then pooled, stained for viability with Calcein AM and Draq7, and quantified using a BD Rhapsody Scanner. Single-cell RNA libraries were prepared using the BD Rhapsody system (BD Biosciences). Cell capture was performed according to manufacturer's instructions. Briefly, single-cell transcriptomics was conducted using the BD Rhapsody Express System, and lysed cells were processed for reverse transcription and exonuclease I treatment. Libraries were created using microbead-captured single cell transcriptomes as per the manufacturer's protocol using the BD Rhapsody cDNA Kit (BD Biosciences, USA) and the BD Rhapsody Targeted mRNA Amplification Kit (BD Biosciences, USA). The BD Rhapsody Immune Response Panel (Cat. # 633753), consisting of 397 genes, and an additional 67 custom genes were used. For sequencing, libraries were quantified using a Qubit Fluorometer (Thermo Fisher Scientific) and KAPA Library Quantification Kit (Roche), adjusted to 2nM, and pooled in a mRNA:sample tag ratio of 12.5:1. Sequencing was performed on an Illumina NextSeq1000.