|
| Status |
Public on Feb 17, 2024 |
| Title |
output-XETG00098__0018582__rep4_atn_post__20231129__015936 |
| Sample type |
RNA |
| |
|
| Source name |
brain hemisection
|
| Organism |
Mus musculus |
| Characteristics |
tissue: brain
genotype: WT
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Brain hemispheres were embedded into OCT and snap frozen in methylbutane cooled by liquid nitrogen after brain removal. Samples were processed according to recommendations in the Xenium In Situ for Fresh Frozen Tissues – Tissue Preparation Guide (10X Genomics): 10 μm anterior (bregma = -0.58mm), intermediate (bregma = -0.70mm to -0.82mm) and posterior (bregma = -0.94mm) hemisections were sectioned using a cryostat and placed within the 12x24mm sample frame on a Xenium slide. Slides were subsequently stored at -80°C until use. For use, slides were fixed and permeabilized as per the Xenium In Situ for Fresh Frozen Tissues – Fixation & Permeabilization protocol (10X Genomics).
|
| Label |
N/A
|
| Label protocol |
N/A
|
| |
|
| Hybridization protocol |
Probe hybridization was performed at 50°C overnight using the pre-designed, 247-probe Xenium Mouse Brain Gene Expression panel (10X Genomics, Cat. #1000462) in conjunction with a custom 50-probe add-on panel (10X Genomics, Cat.#1000646) against select thalamic target genes. Next, slides underwent wash steps to remove any unhybridized probes, followed by a 2-hour ligation step at 37°C to anneal probes to their specific mRNA target. The probes were then enzymatically amplified via rolling circle amplification PCR for 2 hours at 37°C. After washing, quenching steps were performed to reduce background autofluorescence, followed by a DAPI nuclear stain. The slide cassettes were subsequently loaded onto the Xenium Analyzer instrument
|
| Scan protocol |
Following instrument initialization, slides and consumables were loaded onto the Xenium Analyzer, and regions of interest were selected after a preliminary sample scan. Once the Xenium Analyzer run begins, slides undergo iterative rounds of image acquisition where probe-specific fluorescent oligos are added, hybridized to the amplified probe sequence, imaged and gently removed. mRNA-specific puncta were decoded into their corresponding gene IDs and assigned a quality score. Using the DAPI nuclear stain, cell boundaries were segmented and transcripts were spatially assigned to cells at a sub-50nm XY localization precision. After approximately 2 days of imaging and analysis, Xenium data was collected for further analysis.
|
| Description |
fresh frozen single-cell spatial transcriptomics
|
| Data processing |
Xenium In Situ Analyzer performs automated onboard analysis which includes DAPI-based segmentation of nuclei via deep-learning, then expansion of the segmentation masks. A cell-by-gene matrix of segmented nuclei is generated for analysis. Post-processing in R with custom scripts and Seurat
|
| |
|
| Submission date |
Feb 16, 2024 |
| Last update date |
Feb 17, 2024 |
| Contact name |
Mark Steven Cembrowski |
| Organization name |
University of British Columbia
|
| Department |
Cellular and Physiological Sciences
|
| Lab |
Cembrowski
|
| Street address |
2350 Health Sciences Mall
|
| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V6S0B7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL33896 |
| Series (1) |
| GSE255953 |
Transcriptomic landscape of the anterior thalamic nuclei of the mouse [Xenium] |
|
