|
| Status |
Public on Nov 03, 2023 |
| Title |
Midbrain Fzd10 lineage |
| Sample type |
SRA |
| |
|
| Source name |
Dorsal midbrain
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Dorsal midbrain
cell type: Fzd10 lineage
|
| Treatment protocol |
To selectively label and isolate cells of the Fzd10-lineage in the embryonic dorsal midbrain, Fzd10-CreER+/- mice were crossed with mTmG reporter line to generate mTmG;Fzd10-CreER+/- embryos. Timed pregnant females received intraperitoneal (IP) injections of TM (1 mg/mouse dissolved in corn oil) at E10.5. Upon Cre activation, a switch of fluorescent color from red (tdT) to green (GFP), according to the mTmG principle (doi: 10.1002/dvg.20335) was observed in the embryonic midbrain cells. Dorsal midbrain from these mTmG;Fzd10-CreER+/- embryos were isolated at E12.5, E14.5 and E16.5 for scRNA-seq experiments.
|
| Growth protocol |
All animal procedures were approved by the Austrian Federal Ministry of Science and Research in accordance with the Austrian and European Union animal law (license number: BMWF-66.018/0007-II/3b/2012 and BMWFW-66.018/0006-WF/V/3b/2017). Experimental mice were bred and maintained according to regulations approved by institutional animal care and use committee, institutional ethics committee and the guidelines of the preclinical facility (PCF) at IST Austria. Mice were housed at 21±1°C ambient temperature and 40–55% humidity in 12 hrs dark/light cycles. All mouse lines were kept in mixed C57/Bl6, FVB and CD1 genetic background. In some experiments, wild-type CD1 mice were also used. Both male and female littermates of the desired genotypes were used randomly. Mice were used at an age range from 2 – 8 months for breeding and from E9.5 to P30 for experiments. All efforts were made to minimize the number of animals used following the 3R principles.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryonic dorsal midbrains were dissected at E12.5, E14.5 and E16.5 by first cutting the skin and skull to expose the dorsal part of the brain. An incision was made between the forebrain-midbrain boundary and another one between the midbrain-hindbrain boundary with fine surgical scissors. Two horizontal cuts were then made through the aqueduct to free the dorsal midbrains. In total, 9, 8, and 3 dorsal midbrains were pooled for each of E12.5, E14.5 and E16.5 time points, respectively. The subsequent preparation procedures of single-cell suspension from these tissues were adapted and modified from previous protocols (). For details see publication. Single-cell suspensions were incubated with Zombie NIR fixable viability dye (Biolegend) and viable GFP+ cells were sort-purified immediately using a SH800 Cell Sorter (Sony). In total, 33,000 labelled cells were collected from each embryonic time point.
cDNA libraries were generated from pooled embryonic dorsal midbrain samples containing Fzd10-lineage cells using the Chromium Controller and the Next GEM Single Cell 3’ Reagent Kit (v3.1, 10x Genomics) according to the manufacturer’s instructions.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Raw sequening data was processed with the Cell Ranger pipeline (v7.0.0, 10X Genomics) with transcriptome version: mm10-2020-A, including intronic information
Supplementary files format and content: hdf5 file, filtered feature barcode matrix
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| |
|
| Submission date |
Oct 11, 2023 |
| Last update date |
Nov 03, 2023 |
| Contact name |
Florian M Pauler |
| E-mail(s) |
florian.pauler@ista.ac.at
|
| Organization name |
Institute of Science and Technology Austria (IST Austria)
|
| Lab |
Hippenmeyer
|
| Street address |
Am Campus 1
|
| City |
Klosterneuburg |
| ZIP/Postal code |
3400 |
| Country |
Austria |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE245105 |
Multipotent Progenitors Instruct Ontogeny of the Superior Colliculus - pooled embryonic Fzd10 lineage |
| GSE245106 |
Multipotent Progenitors Instruct Ontogeny of the Superior Colliculus |
|
| Relations |
| BioSample |
SAMN37778056 |
| SRA |
SRX22066599 |
