DOI: 10.5061/dryad.08kprr5bx
title: Vizgen MERFISH files for Single-cell analysis reveals M. tuberculosis ESX-1-mediated accumulation of anti-inflammatory macrophages in infected mouse lungs

description: Mycobacterium tuberculosis (MTB) ESX-1, a type VII secretion system, is a
 key virulence determinant contributing to MTB’s survival within lung
 mononuclear phagocytes (MNPs), but its effect on MNP recruitment and
 differentiation remains unknown. Here, using multiple single-cell RNA
 sequencing techniques, we studied the role of ESX-1 in MNP heterogeneity
 and response in mice and murine bone marrow-derived macrophages (BMDM). We
 found that ESX-1 is required for MTB to recruit diverse MNP subsets with
 high MTB burden. Further, MTB induces an anti-inflammatory transcriptional
 signature in MNPs and BMDM in an ESX-1-dependent manner.  Spatial
 transcriptomics revealed an upregulation of anti-inflammatory signals
 within MTB lesions, where monocyte-derived macrophages concentrate near
 MTB-infected cells. Together, our findings suggest that MTB ESX-1
 facilitates the recruitment and differentiation of anti-inflammatory MNPs,
 which MTB can infect and manipulate for survival. Importantly, we provide
 a comprehensive transcriptomic dataset across various models and methods,
 which could contribute to the broader understanding of recruited cell
 heterogeneity during MTB lung infection. This data is associated with
 processed data and code found
 in https://doi.org/10.5061/dryad.0p2ngf28s.
description: <strong>Spatial
 Transcriptomics</strong> Lungs were perfused with
 10 mL of PBS/2 mM EDTA via right ventricle, followed by inflation through
 the trachea with 1mL of optimal cutting temperature (OCT) mixed with PBS
 at 1:1. C57BL/6 mice aerosol infected with H37Rv-zsGreen at ~75 CFU/mouse
 for 28 days were sacrificed. Lungs were embedded in OCT, flash frozen, and
 stored at -80°C for later cryosectioning. To confirm quality, RNA
 integrity number (RIN)126 was obtained by extracting RNA from each block
 (QIAGEN RNeasy Mini Kit, Cat. No. 74104) and analyzing quantity on an
 Agilent Tapestation system. OCT-embedded tissue blocks were later
 cryosection at -20°C to a thickness of 10 μm and mounted onto MERSCOPE
 Slides (Vizgen, PN 20400001). The mounted tissues were immediately fixed
 with 4% PFA at 37°C for 30 minutes, washed with 3x PBS and stored in 70%
 ethanol at 4°C for no more than 1 mo before proceeding following
 the manufacture’s protocol (MERSCOPE) as previously described83 with the
 following exceptions. First, cell boundaries of mounted
 infected tissues including MTB bacterium were stained with the Vizgen Cell
 boundary Staining Kit (Cat. no. 10400009) in conjunction with a polyclonal
 MTB antibody (Abcam, ab905) raised in rabbit diluted to 1:100. After
 washing, the Secondary Staining Mix (Vizgen, PN 20300011) was diluted with
 a blocking solution in two step dilution (1st - 1:100, 2nd - 1:33) to
 ensure correct ratio of primary to secondary staining since a
 custom primary MTB antibody was used.  Imaging: The
 MERFISH gene panel consisted of 122 genes, designed by selecting top
 differentially expressed genes in each cell type from the myeloid scRNA
 sequence atlas, canonical markers, and genes of interest. The probes were
 hybridized, and samples washed prior to gel embedding without deviation
 from manufacter protocol. Samples were then treated with a clearing
 solution and imaged 1 day later per MERSCOPE User Guide 91600001. Only one
 gel-embedded and cleared sample was prepared per imaging run. The
 gel-embedded and cleared sample was washed prior to adding the DAPI / Poly
 T Staining Reagent, and again prior to assembling the onto the MERSCOPE
 Flow Chamber (Vizgen, PN 10300003). Once fully thawed, the MERSCOPE 140
 Gene Imaging Cartridge was activated and a layer of mineral oil was
 carefully added on top of the imaging buffer. The assembled flow chamber
 with sample and imaging cartridge was placed in the MERSCOPE, image mosaic
 was acquired from the regions of interest, and imaging of the sample was
 performed with multiple rounds of hybridization of fluorescent probes
 which resulted in a raw images stack that contained about 1 TB of
 data.
description: # Vizgen MERFISH files for Single-cell analysis reveals M. tuberculosis
 ESX-1-mediated accumulation of anti-inflammatory macrophages in infected
 mouse lungs
 [https://doi.org/10.5061/dryad.08kprr5bx](https://doi.org/10.5061/dryad.08kprr5bx) ## Description of the data and file structure This dataset contains Vizgen MERFISH .vzg images files from a spatial transcriptomics experiment investigating macrophage transcriptional responses in the mouse lung following MTB infection. These files were generated with the Vizgen MERSCOPE and preprocessed using their standard data processing pipeline. This data is linked to the Dryad dataset [https://doi.org/10.5061/dryad.0p2ngf28s, ](https://doi.org/10.5061/dryad.0p2ngf28s)which contains all of the processed count data and additional experimental details.  ### Files and variables #### Files: 202305231432\_TB-CTRL1-MB\_VMSC02401\_region\_0.vzg, 202305231432\_TB-CTRL1-MB\_VMSC02401\_region\_1.vzg, 202305231432\_TB-CTRL1-MB\_VMSC02401\_region\_2.vzg **Description:**  Control mouse lung not infected with MTB, three sections on one slide.  #### Files: 202305251147\_TB-CTRL2-MB\_VMSC02401\_region\_0.vzg, 202305251147\_TB-CTRL2-MB\_VMSC02401\_region\_1.vzg **Description:** Additional control mouse lung not infected with MTB, two sections on one slide.  #### Files: 202309251210\_20230925-RitwicqA-TB\_VMSC02401\_region\_1.vzg, 202309251210\_20230925-RitwicqA-TB\_VMSC02401\_region\_2.vzg **Description:** MTB infected mouse lung from mouse 2, two sections on one slide. #### File: 202305261458\_TB-TEST3-MB\_VMSC02401\_region\_1.vzg **Description:** MTB infected mouse lung from mouse 3, slide 1, region 1.   #### File: 202310091253\_AmandaS-TB-02\_VMSC02401\_region\_0.vzg, 202310091253\_AmandaS-TB-02\_VMSC02401\_region\_1.vzg, 202310091253\_AmandaS-TB-02\_VMSC02401\_region\_2.vzg **Description:** MTB infected mouse lung from mouse 4, 3 sections on one slide.  #### File: 202310111428\_RitwicqA-TB-4b\_VMSC02401\_region\_1.vzg, 202310111428\_RitwicqA-TB-4b\_VMSC02401\_region\_2.vzg **Description:** MTB infected mouse lung from mouse 4, 2 sections on an additional slide.  ## Code/software The code associated with the processing of this dataset for the linked manuscript is available within this dataset and on github at https://github.com/bspeco/ESX1_macrophageheterogeneity_scRNA/releases/tag/v1.0.0 ## Access information This data is associated with processed data and code found in [https://doi.org/10.5061/dryad.0p2ngf28s](https://doi.org/10.5061/dryad.0p2ngf28s)