 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 03, 2023 |
| Title |
Pten KO |
| Sample type |
SRA |
| |
|
| Source name |
Dorsal midbrain
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Dorsal midbrain
cell type: Nestin lineage
|
| Treatment protocol |
To generate MADM genetic mosaic mice for Pten gene, we followed previously established protocols (doi: 10.1016/j.xpro.2021.100939; 10.1016/j.celrep.2021.109274). In brief, the Pten-flox allele was recombined onto Chr.19 containing the MADM-19TG cassette to generate MADM-19TG/TG,Pten-flox stocks. Next, MADM-19TG/TG,Pten-flox were crossed with MADM-19GT/GT;Nestin-Cre+/- to generate Control-MADM (MADM-19GT/TG;Nestin-Cre+/-) and Pten-MADM (MADM-19GT/TG,Pten-flox;Nestin-Cre+/-) mice. Upon Cre recombinase-mediated interchromosomal recombination for reconstitution of fluorescent MADM markers, all (red tdT+, green GFP+ and yellow tdT+/GFP+) cells in Control-MADM were wild-type (Pten+/+) whereas in Pten-MADM red tdT+ cells were wild-type (Pten+/+), green GFP+ cells homozygous mutant (Pten-/-) and yellow tdT+/GFP+ cells heterozygous (Pten+/-) in an otherwise unlabeled Pten+/- environment.
|
| 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 |
To isolate P0 dorsal midbrains, mice were first decapitated and the brains dissected from the skull. After removing the cortices to expose the midbrain, two cuts were made with sharp razor blades: one between the forebrain-SC boundary and another between the SC-inferior colliculus boundary. Both cuts were angled to meet at the aqueduct thus avoiding contamination from ventral midbrain regions. In total, 26 Control-MADM and 24 Pten-MADM dorsal midbrains were separately pooled. The subsequent preparation procedures of single-cell suspension from these tissues were adapted and modified from previous protocols (10.1016/j.xpro.2020.100215). 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, 14,000 and 12,000 labelled cells were sorted from P0 time point of Control-MADM and Pten-MADM, respectively.
cDNA libraries were generated from Control-MADM, Pten-MADM dorsal midbrain samples using the Chromium Controller and the Next GEM Single Cell 3’ Reagent Kit (v3.1, 10x Genomics) according to the manufacturer’s instructions.
|
| |
|
| 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
|
| |
|
| 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) |
| GSE245104 |
Multipotent Progenitors Instruct Ontogeny of the Superior Colliculus – Pten |
| GSE245106 |
Multipotent Progenitors Instruct Ontogeny of the Superior Colliculus |
|
| Relations |
| BioSample |
SAMN37778039 |
| SRA |
SRX22066541 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7836813_Pten_MBd_KO_filtered_feature_bc_matrix.h5 |
22.9 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
