GSM8721086: Exp1, Splenic B cells, Day7, scRNAseq, mRNA; Mus musculus; RNA-Seq
Spleens were harvested from mice and homogenised through 70µM cell strainers. RBCs were lysed using Pharm Lyse (BD) for 3 minutes at room temperature and then single cell suspensions washed twice in 1x PBS. Samples were then incubated with Fc receptor-blocking antibodies (BD) for 20 minutes on ice, followed by incubation with titrated monoclonal fluorescently conjugated antibodies and TotalSeq-C (BioLegend) mouse hashtags for 20 minutes on ice. Cells were washed twice and resuspended with propidium iodide (1:500 dilution) in PBS 2% BSA. For experiment 1, samples from five individual mouse replicates were pooled together for each timepoint. Note, for timepoint day 4 post infection, there were four separately infected mice with one uninfected naive sample pooled in. Live, TCRβ negative, CD19+, B220int-hi cells were sorted using a FACS Aria III (BD) into a 2% BSA/PBS buffer. For experiment 2, samples from seven individual mice (three for PcAS, three for PcAS+drug and one uninfected) were pooled together for each timepoint. Live, TCRβ negative, CD19+, B220int-hi, IgDlo cells were sorted using a FACS Aria III (BD) into a 2% BSA/PBS buffer. For each sample, a small number of IgDhi naive cells were spiked in to assist in removal of batch effects for downstream scRNAseq analysis. After sorting, cells were loaded onto the chromium controller and using the Chromium GEM-X Single Cell 5' Kits v3 (Catalogue #1000695;10X Genomics), cDNA-sequencing libraries were prepared as per manufacturer's instructions.