Validation of the 5’Citeseq assay to characterize cell surface protein expression on single cells 

Background: CITE-seq is a scalable method that uses oligonucleotide-tagged antibodies to integrate multiplexed cell surface protein, unbiased transcriptome measurements and immune profiling into a single-cell read-out to give detailed analyses of cell phenotypes. Methods:  Chromium 10X 5’ sc-RNA-CITE-seq experiments using single-cell suspensions from thawed healthy donor PBMCs both unstimulated and subjected to SEB stimulation and blister fluid cells from a patient with an acute severe cutaneous blistering disease were selected magnetically for live cells using Annexin V beads. 5’ Cell hashing using DNA-sequence tagged antibodies was utilized to test pooling of samples in a single 10X reaction. Suspensions were stained with 5’ DNA-sequence tagged antibodies (TotalSeq-C) that represented standard lymphocyte lineages (CD3, CD4, CD8, CD14, CD16, CD19, CD20, CD27, CD45RO, CD45RA, CCR7, CD95, CD127, CD56).  Antibodies were titrated to 0.5x, 1x and 2x concentrations under stimulated and unstimulated conditions, 1x for the blister fluid and a pooled sample including all concentrations. CellRanger (version 3.0.0) was used to demultiplex the raw sequencing data and extract filter and correct barcodes and unique molecular identifiers (UMIs), remove cDNA PCR duplicates and align reads to the human transcriptome (GRCh38) using STAR. The resulting BAM files and filtered count matrices were used in downstream analyses in visual genomics analysis studio (VGAS). Results:  Reproducible results were obtained with antibody titrations 0.5x suggesting that lower concentration should be feasible and trialed. 5’cell hashing is a viable approach for pooling samples for CITE-seq experiments.  CITE-seq results were comparable to flow cytometry providing further validation.  VGAS is a user-friendly visualization and data analysis platform suitable for these analyses. Conclusions:   We have confirmed that 5’CITE-seq with cell hashing is a scalable and cost-effective approach for defining cell phenotypes from a variety of sample types under differing conditions.