Abstract
Single cell sequencing has recently been applied to many immunological studies. Flow cytometric index sorting isolates cells for single cell sequencing with protein level data linked to sequences. However, successful sequencing of index sorted samples requires careful optimization of several sort parameters, including nozzle size, flow rate, threshold rate, and yield calculations. In this study, considerations and optimization data for each of these variables are presented. Our analysis focused on index sorting, but the findings can be applied to any plate sorting protocol. Minimization of flow rates and use of the 70 μm nozzle improved cell yields. Improvements in total read counts after sequencing were obtained by decreasing the threshold rate, or the number of cells processed per second. In addition, this technique provided linked protein and gene expression analysis of the cytokine interferon (IFN)γ, demonstrating that on a single cell basis IFNγ+ cells tend to express IFNG mRNA, and IFNγ- cells do not. Through rigorous optimization and quality control, we have identified parameters important to plate sorting and recommend the use of the 70 μm nozzle and low flow and threshold rates for analysis of rare populations of human lymphocytes.