GSM9228167: Rhesus macaque spleen CD45+ cells, multiplexed run 1; Macaca mulatta; RNA-Seq
Spleen mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation (Sigma). Cell suspensions were stained with a CD45 antibody. Four samples (Naive, Infected, and two treated RMs) were pooled and loaded onto a single BD Rhapsody cartridge (~40,000 cells per cartridge). Cells were lysed and polyadenylated mRNAs were captured on barcoded magnetic beads according to the BD Rhapsody protocol. Each transcript was tagged with a unique molecular identifier (UMI) and a cell-specific barcode. cDNA was synthesized and amplified following the manufacturer's instructions prior to library preparation Single-cell cDNA libraries were generated using the BD Rhapsody Single-Cell RNA-Seq Library Preparation Kit according to the manufacturer's instructions. After reverse transcription and second-strand synthesis, cDNAs containing unique molecular identifiers (UMIs) and cell barcodes were amplified by PCR. Sequencing libraries were prepared following the BD Rhapsody workflow, including fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR. Library quality and size distribution were assessed using the Agilent 4200 TapeStation, and concentrations were measured with the Qubit dsDNA HS assay kit (Invitrogen). Pooled libraries were sequenced on an Illumina NovaSeq 6000 platform with paired-end reads, targeting a depth of ~50,000 reads per cell and ~600 reads per cell for sample tag libraries.