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Status |
Public on Jun 21, 2023 |
Title |
replicate 1, MERFISH |
Sample type |
RNA |
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Source name |
MERFISH
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Organism |
Mus musculus |
Characteristics |
cell type: brain cells tissue: Brain strain: C57BL/6J age: 18 weeks molecule: other instrument model: Vizgen
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Treatment protocol |
Lysophosphatidylcholine (LPC) injections were administered at the age of 16-18 weeks. A solution of 1% LPC (L4129, Sigma) in PBS was mixed with Monastral blue (274011, SigmaAldrich) at a concentration of 0.03% to aid with visualization of the lesion during tissue processing. Mice were anaesthetized with an intraperitoneal injection of MMF solution (0.5 mg medetomidin/kg (body weight), 5.0 mg midazolam/kg (body weight) and 0.05 mg fentanyl/kg (body weight)). Then, head fur was removed, the eyes were treated with bepanthene cream (1578847, Bayer) and a small incision in the skin was performed to expose the skull. The mouse was positioned into a stereotactic injection apparatus and a small hole was drilled at the following injection coordinates (from bregma): X, ± 1.0 mm; Y, −0.1 mm). A glass capillary containing the LPC–monastral blue solution was then lowered to Z: −1.30 mm from bregma, and 1 µL was injected at a rate of 100 nL/minute. Two minutes after the delivery of LPC, the capillary was slowly retracted. The mouse was then injected with 0.05 mg buprenorphin/kg (body weight), and the skin was sutured. Anesthesia was terminated by a subcutaneous injection of AFN solution, containing 2.5 mg/kg (body weight) atipamezol, 1.2 mg/kg (body weight) naloxon and 0.5 mg/kg (body weight) flumazenil.
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Extracted molecule |
total RNA |
Extraction protocol |
18 days after LPC injection, mice were anaesthetized with an i.p. injection of MMF (fentanyl (0.05 mg/kg)–midazolam (5 mg/kg)–medetomidine (1 mg/kg)) and transcardially perfused with 2 UI/mL Heparin (Heparin-Natrium-25000-ratiopharm®, PZN: 03029843) in HBSS (no calcium, no magnesium, Gibco™, 14175129) for 3 min and 4 % paraformaldehyde (PFA, EM Grade, Electron Microscopy Sciences, Cat. No. 15710, diluted in 10X PBS, Invitrogen™, AM9624 and UltraPure™ Distilled Water, Invitrogen™, 10977-035) for 5 min, before carefully removing the brain from the skull. Afterwards, the brains were fixed by submergingin 4% PFA for 6 hours, followed by 14 hours in 15% sucrose (Sigma-Aldrich, S0389 in UltraPure™ Distilled Water, Invitrogen™, 10977-035) and 5 hours in 30% sucrose. Next, the PFA-fixed brains were simultaneously embedded in Tissue-Tek® O.C.T.™ Compound (Sakura, 4583) and frozen in a plastic mold on dry ice. For the fresh frozen brain samples, the mouse was only perfused with Heparin in HBSS and directly embedded and frozen in Tissue-Tek® O.C.T.™ Compound on dry ice. Brains were stored at -80°C until further processing. Coronal, 10 µm thick brain sections were prepared and collected at a cryotome (CryoStar NX70, Thermo Scientific). Sections determined for MERFISH analysis were placed on round glass slides provided by Vizgen Corp. (Cambridge, MA 02138) and functionalized in-house by coating one side with a 0.1 mg/mL poly-L-ornithine solution (Sigma-Aldrich, P4957 in PBS) for 1 hour, before drying them. Brain sections for MERFISH were subsequently washed two times with PBS and one time with 70 % ethanol (VWR Chemicals, 20.821.310, diluted in UltraPure™ Distilled Water, Invitrogen™) for 5 minutes each. Then samples were individually sealed in bags filled with 70 % ethanol and shipped to Vizgen Corp. (Cambridge, MA 02138) for MERFISH analysis. MERFISH Brain sections were measured by MERFISH according to manufacturer's protocol (https://vizgen.com/technology/#merfish)
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Label |
n/a
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Label protocol |
n/a
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Hybridization protocol |
n/a
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Scan protocol |
n/a
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Description |
panel of 287 genes was measured by MERFISH
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Data processing |
Encoded MERFISH images were processed according to manufacturer's pipeline (https://vizgen.com/technology/#merfish). The output of this pipeline are spatial positions of individual transcripts and segmented single cells based on DAPI and poly-A stainings. Single cell expression tables are produced by counting individual transcripts within segmented cell boundaries. Tab-separated single cell expression matrix and cell metadata
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Submission date |
May 10, 2022 |
Last update date |
Jun 21, 2023 |
Contact name |
Peter Androvic |
Organization name |
LMU Hospital, Institute for Stroke and Dementia Research
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Lab |
Gokce Lab
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Street address |
Feodor-Lynen-Strasse 17
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City |
Munich |
State/province |
Bavaria |
ZIP/Postal code |
81377 |
Country |
Germany |
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Platform ID |
GPL31217 |
Series (2) |
GSE202623 |
Spatial Transcriptomics correlated Electron Microscopy [MERFISH] |
GSE202638 |
Spatial Transcriptomics correlated Electron Microscopy |
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Supplementary file |
Size |
Download |
File type/resource |
GSM6125919_b2s20_cell_by_gene.csv.gz |
11.6 Mb |
(ftp)(http) |
CSV |
GSM6125919_b2s20_cell_metadata.csv.gz |
7.4 Mb |
(ftp)(http) |
CSV |
GSM6125919_b2s20_detected_transcripts.csv.gz |
1.1 Gb |
(ftp)(http) |
CSV |
Processed data provided as supplementary file |
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