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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Aug 01, 2024 |
| Title |
Postnatal day 21 (P21) normoxia, biological rep 2 |
| Sample type |
RNA |
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| Source name |
Whole brain, perinatal normoxia
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| Organism |
Mus musculus |
| Characteristics |
tissue: Whole brain
treatment: perinatal normoxia
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| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen brain were cryo-sectioned at 10 μm thickness at -21°C, and mounted onto MERSCOPE beaded coverslips. Sections were allowed to refreeze, and then fixed with 4% paraformaldehyde, diluted in 1x PBS for 15 minutes, washed with 1x PBS for 5 minutes each, then stored in 70% ethanol overnight.
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| Label |
Gene targeting probes
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| Label protocol |
Single-cell spatial transcriptomics was performed using a gene panel targeting 497 transcripts.
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| Hybridization protocol |
A total of 497 genes were selected and assigned a 20-bit binary barcode which was used for generating MERFISH probes. Sections were washed with 1x PBS followed by Sample Prep Wash Buffer (Vizgen, PN 20300001). Sections were incubated in Formamide Wash Buffer (Vizgen, PN 20300002) for 30 min at 37°C, then incubated in the gene panel mix for 42-46 hr at 37°C. Sections were then incubated two times in Formamide Wash Buffer for 30 min each at 47°C, and washed with sample prep wash buffer for at least 2 min. Sections were coated in gel embedding solution (0.05% w/v ammonium persulfate, 0.05% v/v N,N,N’,N'-tetramethylethylenediamine in Gel Embedding Premix (Vizgen, PN 20300004)) and incubated at RT for 1.5 hr, cleared in 1:100 proteinase K in Clearing Premix (Vizgen, PN 20300003) at 37°C overnight or for a maximum of 7 days to clear lipids and proteins that may contribute to auto-fluorescence background noise. Prior to imaging, sections were washed two times with Sample Prep Wash Buffer, incubated in DAPI and PolyT Staining Reagent (Vizgen, PN 20300021) for 15 min on a rocking platform, incubated in Formamide Wash Buffer for 10 min, and washed again with Sample Prep Wash Buffer.
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| Scan protocol |
Imaging was performed on the MERSCOPE platform (Vizgen, Cat: 10000001) according to manufacturer instructions.
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| Description |
mouse brain gene panel
NX2
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| Data processing |
Transcript barcodes were read and matched assigned to the correct gene. MERFISH images were segmented using Vizgen's post-processing tool and Cellpose v2. DAPI and PolyT signals were used to delinate the cell boundaries. Individual RNA molecules were assigned to a cell based on whether they were positioned within the marked boundary. Anatomical segmentation was performed using the MERSCOPE Visualizer software. Cells for MERFISH were processed using the Seurat V5 package in R.
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| Submission date |
Feb 15, 2024 |
| Last update date |
Aug 01, 2024 |
| Contact name |
Brian Kalish |
| E-mail(s) |
brian.kalish@sickkids.ca
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| Phone |
6305329007
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| Organization name |
Harvard Medical School
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| Department |
Neurobiology
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| Lab |
Greenberg Lab
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| Street address |
220 Longwood Ave
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL31217 |
| Series (1) |
| GSE255892 |
Perinatal Brain Injury Triggers Niche-Specific Changes to Cellular Biogeography [MERFISH] |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM8082303_NX2_cell_by_gene.csv.gz |
23.2 Mb |
(ftp)(http) |
CSV |
| GSM8082303_NX2_cell_metadata.csv.gz |
54.5 Mb |
(ftp)(http) |
CSV |
| GSM8082303_NX2_detected_transcripts.csv.gz |
1.7 Gb |
(ftp)(http) |
CSV |
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