single-cell RNA-seq of Merkel cell carcinoma
Cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed, labeled, and prepared for 10x droplet encapsulation following a modified protocol adapted from the 10x and BioLegend TotalSeq C protocols. Cells were blocked using Human TruStain FcX™ Fc blocking reagent (BioLegend) and stained with TotalSeq™ C and hashing antibodies (BioLegend). Live cells were sorted using fluorescence-activated cell sorting (FACS) with a gate set for CD45+ and CD3+ cells. The sorted cells were combined with CD3- cells to maintain a 1:3 ratio and cryopreserved in Bambacker preservation medium with SUPERase•In™ RNase Inhibitor (Sigma). Equal volumes of four to five hashed samples were combined to ensure that samples stained with the same hashing antibody were not mixed. The cells were slow-frozen at -80°C before being transferred to liquid nitrogen for long-term storage. Single-cell ADT libraries were prepared using the 10x Genomics Single cell ADT protocol.