|
| Status |
Public on Mar 14, 2023 |
| Title |
OB_PBS_2 |
| Sample type |
RNA |
| |
|
| Source name |
Olfactory bulb
|
| Organism |
Mus musculus |
| Characteristics |
genotype: C57BL6/J
Sex: Female
age: P53
tissue: Olfactory bulb
treatment: PBS inoculation
timepoint: 24 hours after fifth inoculation
|
| Treatment protocol |
Brains were isolated from P53 mice following five repeated inoculations with either a suspension of Group A Streptococcus or with vehicle (PBS), and profiled at 24 hours post-inoculation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After perfusion with cold PBS, brains were embedded in OCT for sectioning onto specialized coverslips. 10 microns coronal sections of the olfactory bulb (around bregma 4.28) were collected for analysis by MERFISH
|
| Label |
N/A
|
| Label protocol |
Tissue on coverslips was fixed for 15 minutes at room temperature in 4% paraformaldehyde in PBS, followed by three washes with PBS. Tissue was then permeabilized in 70% ethanol for 24 hours. After a wash with Formamide Wash Buffer (30% formamide in 2X saline sodium citrate (SSC)), the MERFISH library mix was added and allowed to hybridize to occur for 48 hours. Sample was then washed and incubated at 47°C with Formamide Wash Buffer twice and then the tissue was embedded in a polyacrylamide gel followed by incubation with tissue clearing solution (2X SSC, 2% SDS, 0.5% v/v Triton X-100, and proteinase K 1:100) overnight at 37°C.
|
| |
|
| Hybridization protocol |
Tissue was washed and hybridized for 15 minutes with the first hybridization buffer containing readout probes associated with the first round of imaging as well as the reagents for DAPI and polyT staining.
|
| Scan protocol |
MERFISH imaging was performed as previously described (Moffitt et al., 2018) with parameters files provided by Vizgen. Briefly, the sample was loaded into a flow chamber connected to the Vizgen Alpha Instrument. First, a low-resolution mosaic image was acquired (405 nm channel) with a low magnification objective (10x). Then the objective was switched to a high magnification objective (60x) and seven 1.5 μm z-stack images of each field of view position were generated in 749 nm, 638 nm and 546 nm channels. A single image of the fiducial beads on the surface of the coverslip was acquired and used as a spatial reference (477 nm channel). After each round of imaging, the readout probes were extinguished, and the sample was hybridized with the next set of readout probes. This process was repeated until combinatorial FISH was completed.
|
| Description |
CountsAndCellMetadata_Coverslip_2.csv
PBS_2 sample is at positions <5600 Y
|
| Data processing |
Raw data were decoded using the MERlin pipeline (Vizgen, v0.1.6 ) using the relevant library codebook. Cell boundaries were segmented in each FOV using a seeded watershed algorithm using DAPI signal as the seed and poly-T signal as the watershed channel. The volume, X position, and Y position of these cell boundaries, as well as the probe counts within each cell boundary, were output to CountsAndCellMetadata.csv files for further analysis. MERFISH data was analyzed in RStudio using Seurat and custom-made scripts as previously described (Feinberg et al 2022). Cell segmentations with volume < 50µm3 or < 10 unique transcripts were first excluded. Cell gene expression data of each cell was then normalized to that cell’s volume and the total transcript count of that cell, then scaled. To correct for global differences in total transcript counts between coverslips (each containing 1 GAS sample and 1 PBS sample), we performed ComBat batch correction (sva 3.38.0). To identify individual cell types, we performed principle component analysis was performed using the entire probe library (391 transcripts) as the variable features, followed by linear dimensional reduction. Clusters were manually annotated based on the spatial distribution of the cells in the tissue and the expression cell type-specific marker genes. Because of imperfections in cell boundary segmentation, a small fraction of cells expressed cell type markers for multiple cell types. Clusters composed of these “hybrid” cells were removed from the analysis, and UMAP embedding and clustering analysis were iteratively repeated until all “hybrid” clusters were removed. Nearest neighbor analysis of microglial distance to T cells was calculated based the X, Y coordinates of the centers of the cell segmentations using a custom python script.
CountsAndCellMetadata.csv files contain matrices of cell IDs as rows, genes and metadata as columns. Matrix contains raw counts of detected transcripts. Volume metadata is provided in cubic microns. X and Y metadata are provided in microns.
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| |
|
| Submission date |
Dec 15, 2022 |
| Last update date |
Mar 14, 2023 |
| Contact name |
Travis E. Faust |
| E-mail(s) |
travis.faust@umassmed.edu
|
| Organization name |
University of Massachusetts Chan Medical School
|
| Department |
Neurobiology
|
| Lab |
Schafer
|
| Street address |
364 Plantation Street
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01655-0002 |
| Country |
USA |
| |
|
| Platform ID |
GPL31217 |
| Series (1) |
| GSE221106 |
Spatial analysis of microglial and vascular responses following recurrent intranasal Group A Streptococcus infections |
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