Protocol for investigating intracellular microbial diversity using single-cell RNA-seq in immune cells of SARS-CoV-2-positive and recovered patients.

Intracellular microorganisms like viruses and bacteria impact immune cell function. However, detection of these microbes is challenging as the majority exist in a non-culturable state. This protocol presents detailed steps to investigate intracellular microbial diversity using single-cell RNA sequencing (scRNA-seq) in immune-cells of SARS-CoV-2-positive and recovered patients. We present a workflow from sample collection to library preparation, covering peripheral blood mononuclear cell (PBMC) isolation, single-cell labeling, cartridge priming, and cell lysis. We outline the steps for analyzing the scRNA-seq data, from data quality control (QC) to detection of intracellular microbes. For complete details on the use and execution of this protocol, please refer to Yadav et al.¹.