Protocol for multi-modal single-cell RNA sequencing on M. tuberculosis-infected mouse lungs

Area of Science:

• Immunology and Microbiology
• Single-cell genomics
• Infectious disease research

Background:

• Understanding the host immune response to Mycobacterium tuberculosis (Mtb) is crucial for controlling tuberculosis.
• Existing methods lack the ability to comprehensively analyze individual infected immune cells.
• Lung macrophages play a key role in Mtb infection dynamics.

Purpose of the Study:

• To develop and describe a novel multi-modal single-cell RNA sequencing (scRNA-seq) protocol.
• To simultaneously assess the transcriptome, surface marker expression, and bacterial phenotype of Mtb-infected lung macrophages.
• To enable a deeper understanding of immune cell contributions to Mtb infection control or progression.

Main Methods:

• Development of a protocol for multi-modal scRNA-seq on Mtb-infected lung macrophages.
• Inclusion of steps for sorting infected cells, CITE-seq antibody staining, methanol fixation, and scRNA-seq library generation.
• Adaptability of the protocol for use with tissues from murine, nonhuman primate, and human infections.

Main Results:

• Successful implementation of a protocol that integrates transcriptome, surface protein, and bacterial phenotype data at the single-cell level.
• Demonstration of the protocol's utility in analyzing Mtb-infected macrophages.
• Establishment of a versatile technique applicable across different species.

Conclusions:

• The developed multi-modal scRNA-seq protocol provides a powerful tool for dissecting complex host-pathogen interactions in Mtb infection.
• This technique facilitates a more nuanced understanding of how individual immune cells influence tuberculosis disease progression.
• The protocol's cross-species applicability broadens its potential impact on infectious disease research.