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使用单细胞RNA测序研究新冠病毒阳性和康复患者免疫细胞中细胞内微生物多样性的方案。

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

作者信息

Soni Jyoti, Mehta Priyanka, Yadav Sunita, Chattopadhyay Partha, Pandey Rajesh

机构信息

Division of Immunology and Infectious Disease Biology, INtegrative GENomics of HOst-PathogEn (INGEN-HOPE) Laboratory, CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB), Mall Road, Delhi 110007, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.

Division of Immunology and Infectious Disease Biology, INtegrative GENomics of HOst-PathogEn (INGEN-HOPE) Laboratory, CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB), Mall Road, Delhi 110007, India.

出版信息

STAR Protoc. 2025 Mar 21;6(1):103546. doi: 10.1016/j.xpro.2024.103546. Epub 2025 Jan 8.

DOI:10.1016/j.xpro.2024.103546
PMID:39786999
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11780099/
Abstract

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..

摘要

像病毒和细菌这样的细胞内微生物会影响免疫细胞功能。然而，检测这些微生物具有挑战性，因为大多数微生物处于不可培养状态。本方案介绍了在新冠病毒阳性和康复患者的免疫细胞中使用单细胞RNA测序（scRNA-seq）研究细胞内微生物多样性的详细步骤。我们展示了从样本采集到文库制备的工作流程，包括外周血单核细胞（PBMC）分离、单细胞标记、芯片灌注和细胞裂解。我们概述了分析scRNA-seq数据的步骤，从数据质量控制（QC）到细胞内微生物的检测。有关本方案使用和执行的完整详细信息，请参考亚达夫等人的研究。

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