title: GSM4548078: Vehicle-treated naïve mice (0 dpi); Mus musculus; RNA-Seq
library_construction_protocol: Peripheral blood leukocytes were collected from AV or vehicle-treated mice (uninfected or 1 dpi) from three biological replicates per group and pooled. Leukocytes were subjected to CD4/CD8/CD19 depletion. Leukocytes of the peripheral blood were isolated following two rounds of ACK lysis. T and B cells were depleted using CD4, CD8, and CD19 microbeads (Millitenyi) to enrich for myeloid cells of the peripheral blood. Cells were resuspended at 100 cells/μL in PBS containing 0.04% (w/v) BSA to minimize cell loss and aggregation. ∼17,500 cells were partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs) to achieve single cell resolution for a maximum of 10,000 individual cells per sample. For gene expression libraries (GEX), Isolated single cell suspensions were subjected to droplet-based massively parallel single-cell RNA sequencing using Chromium Single Cell 3' v2 Reagent Kit in a BSL-3 level laboratory as per manufacturer's instructions (10x Genomics). Briefly, cell suspensions were loaded at 1,000 cells/uL with the aim to capture 10,000 cells/well. The 10x Chromium Controller generated GEM droplets, where each cell was labeled with a specific barcode, and each transcript labeled with a unique molecular identifier (UMI) during reverse transcription. The barcoded cDNA was isolated via a Dynabeads MyOne SILANE bead cleanup mixture and amplified 8 cycles. Amplified cDNA was purified via SPRI bead cleanup and removed from the BSL-3 space for library generation.  Amplified cDNA underwent fragmentation, end repair, A-tailing, adapter ligation and sample index PCR as per the manufacturer's instructions to generate gene expression libraries. Libraries were sequenced on a NovaSeq S2 (200 cycle) flow cell, targeting 50,000 read pairs/cell.
attributes.tag: GEO Accession
attributes.value: GSM4548078