Comparing the impact of sample multiplexing approaches for single-cell RNA-sequencing on downstream analysis using cerebellar organoids.

Multiplexing overcomes limited throughput in single-cell RNA sequencing (scRNA-seq). Commercial strategies include Parse Biosciences combinatorial barcoding (Parse) and 10x Genomics CellPlex with microfluidic capture (10x). It is currently unknown how these techniques differ when characterizing complex tissues. Cerebellar organoids are a highly relevant model for studying cerebellar evolution, development, and disease. Yet, their extensive characterization through scRNA-seq is ongoing. Therefore, we compared the two multiplexing techniques using cerebellar organoids. While both strategies demonstrated technical reproducibility and revealed comparable cellular diversity, we found more stressed cells in 10x than in Parse. Additionally, Parse covered a higher gene biotype diversity and showed lower mitochondrial and ribosomal protein-coding transcript fractions. In summary, we demonstrate that both techniques provide similar insight into cerebellar organoid biology, but the flexibility of experimental design, capture of long transcripts, and the level of cell stress caused by the two workflows differ.