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. 2020 Oct 6;1(3):100123.
doi: 10.1016/j.xpro.2020.100123. eCollection 2020 Dec 18.

Dual RNA-Sequencing of Mycobacterium tuberculosis-Infected Cells from a Murine Infection Model

Davide Pisu  1 , Lu Huang  1 , Bom Nae Rin Lee  1 , Jennifer K Grenier  2 , David G Russell  1
Affiliations

Dual RNA-Sequencing of Mycobacterium tuberculosis-Infected Cells from a Murine Infection Model

Davide Pisu et al. STAR Protoc. .

Abstract

Dual RNA-sequencing is a powerful technique to assess both bacterial and host transcriptomes in an unbiased way. We developed a protocol to perform Dual RNA-seq on in vivo-derived macrophage populations infected with Mycobacterium tuberculosis. Here, we provide a practical step-by-step guide to execute the protocol on Mtb-infected cells from a murine infection model. Our protocol can also be easily applied to perform Dual RNA-seq on in vitro-derived cells as well as different Mtb-infected host cell types. For complete details on the use and execution of this protocol, please refer to Pisu et al. (2020).

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Generation of a Single-Cell Lung Suspension (A) Mouse is sprayed with 70% Ethanol. (B) Skin is removed and chest walls exposed. (C) Removal of the chest walls to access the lobes of the lung. (D) Lobes are removed. (E) GentleMACS C Tube with the lung in the dissociation solution. (F) Manual dissociation of the mouse lung in a 6 well-plate. (G) Lungs are minced with scissors. (H) Minced lungs. (L) The lung is transferred to a 70uM cell strainer, using a wide-bore pipette tip. (M) Lungs are grinded against the mesh of the cell strainer.
Figure 2
Figure 2
Resuspending the Bacterial/Cell Debris Pellet in Trizol and Zirconia Beads (A) ~150ul of Trizol supernatant is left behind in the tube containing the bacterial/cell debris pellet. (B) 150ul of Zirconia Beads and 400ul of fresh Trizol are added to the same tube.
Figure 3
Figure 3
Examples of a Fragment Analyzer Track (A) In the top right corner is the expected result of the Fragment Analyzer track when following this protocol. RQN values <6 may lead to inefficient rRNA removal when using the Ribo-Zero or similar kits. (B) Example of a Bioanalyzer track of the final library from one of our low input dual RNA-seq samples.
See this image and copyright information in PMC

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