Toward Cell Type-Specific In Vivo Dual RNA-Seq.

Area of Science:

• Microbiology
• Genomics
• Infectious Diseases

Background:

• Dual RNA-sequencing (RNA-seq) profiles gene expression in both host and pathogen simultaneously.
• Existing methods lack host cell-type resolution in in vivo infection models.
• Salmonella Typhimurium is a significant enteric pathogen causing significant disease burden.

Purpose of the Study:

• To develop a detailed protocol for cell type-specific Dual RNA-seq in murine colonocytes.
• To enable simultaneous gene expression profiling of Salmonella Typhimurium and host colonocytes in vivo.
• To overcome challenges of low RNA input in in vivo transcriptomics.

Main Methods:

• Isolation and fixation of murine colonocytes from infected mice.
• Fluorescence-activated cell sorting (FACS) to isolate Salmonella-invaded colonocytes.
• Ribosomal RNA depletion and library preparation for ultralow RNA input.
• Comparison of different library preparation protocols.

Main Results:

• Successful application of Dual RNA-seq to isolated murine colonocytes.
• Demonstration of an ultralow input protocol for in vivo transcriptomics.
• Identification of challenges and advantages of various library preparation methods for minute RNA amounts.

Conclusions:

• The developed protocol facilitates cell type-specific in vivo Dual RNA-seq.
• This method allows charting pathogen gene expression within its niche and the host response.
• The ultralow input protocol holds promise for advancing in vivo host-pathogen interaction studies.