Organ models have received widespread attention in the study of SARS-CoV-2, the pathogen causing the current COVID-19 pandemic. Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human lung organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed

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... on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human lung organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), as an exemplary host factor of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human lung organoids and the subsequent RT-qPCR to quantify the expression level of e.g., ACE2or other host factors of SARS-CoV-2 on RNA level. Protocol set 3 contains an in-depth explanation on how to infect human lung organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human lung organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human lung organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human lung organoids based on material from different donors.