Stepwise single-cell-resolved deep immunophenotyping pipeline to characterise immune heterogeneity and functionality in health and disease

Providing comprehensive insights into both immune cellular compositions and molecular profiles at single-cell resolution remains challenging. Here we proposed a stepwise single-cell-resolved deep immunophenotyping (SCRIPT) pipeline to enable not only a high-dimensional characterization of the immune compositions and functionalities, but also an unbiased genome-scale molecular profiling of specific immune subsets. Our pipeline integrated rigorous pre-analytical measures, including consideration of circadian timing during sample collection, multiple quality control steps, and critical parameters at different stages of the workflow. Our pipeline leveraged the first-step cytometry analysis to rationally guide subsequent efficient and unbiased single-cell RNA sequencing characterization on specific cell subsets, which otherwise has to be achieved by trial and error and at a much higher cost. We exampled our pipeline with specifically affected CD8 T-cell subsets and a guided genome-scale molecular analysis into those CD8 subsets in a clinical study. Together, our pipeline could be generally applicable to various disease contexts for a comprehensive analysis at both molecular and cellular levels.