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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 09, 2023 |
| Title |
AAV-GFAP-Gq-DREADD_107 |
| Sample type |
SRA |
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| Source name |
Hippocampus and Cortex
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
tissue: brain
group: Saccharin
genotype: Gq-DREADD
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| Treatment protocol |
Mouse samples 104 and 105 were given CNO in their drinking water for 8 weeks, while sampples 106 and 107 were given saccharin in their drinking water
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
8-week old C57BL/6J male mice were injected the AAV-GFAP-hM3D(Gq)-mCherry virus in the hippocampus and cortex using a stereotaxic instrument. They were given either CNO or Saccharin for 8 weeks in their drinking water. Upon tissue collection, the mice were euthanized and perfused using ice-cold PBS. The hippocampal and cortical tissue containing the virus was dissected, minced with a razor blade and incubated for 30 min in ice-cold Accutase at 4 degrees C. The dissociated tissue was centrifuged at 300g for 10 min at 4°C. The pellet was resuspended in ice-cold Hanks’ Balanced Salt solution (Life Technologies, Cat. No. 14025-092) and triturated with a 15 mL serological pipette. This step was repeated until the entire tissue was dissociated. Next, the sample was filtered first through a 70 μm cell strainer, followed by a 40 μm cell strainer. The cells were centrifuged at 300g for 5 min at 4°C. Myelin removal was performed following the Miltenyi’s Myelin Depletion Protocol. In short, the pellet obtained during cell isolation was resuspended in magnetic-activated cell sorting (MACS) buffer (0.5% BSA in PBS, Thermofisher, Cat. No. AM2618), myelin removal beads (Miltenyi Biotec, Cat. No. 130-096-733) were added, and the solution was incubated for 15 min at 4°C. The cells were centrifuged at 300g for 10 min at 4°C, the pellet was resuspended in MACS buffer and the solution was passed through LS columns (Miltenyi Biotec, Cat. No. 130-042-401). The cells were centrifuged at 300g for 15 min at 4°C, the pellet was resuspended in MACS buffer, centrifuged again at 300g for 15 min at 4°C and resuspended in PBS containing 0.01% BSA. The obtained single cells were subjected to droplet-based single cell RNA sequencing. The 10x Genomics ChromiumTM Single Cell 3’ Library & Gel Bead Kit v2 was used per manufacturer’s instructions. The libraries were sequenced on an Illumina NovaSeq 6000 SP.
10x Genomics ChromiumTM Single Cell 3’ Library & Gel Bead Kit v2 protocol
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
single cell RNA sequencing
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| Data processing |
quality control, Cell Ranger Single Cell Software Suite (v.3.0.2)
barcode processing, Cell Ranger Single Cell Software Suite (v.3.0.2)
single-cell 3' gene counting, Cell Ranger Single Cell Software Suite (v.3.0.2)
alignment, Cell Ranger Single Cell Software Suite (v.3.0.2)
Genome_build: mm10
Supplementary_files_format_and_content: Filtered matrix of gene by cell expression, file with barcodes and file with expressed genes. Files are ready to be imported to Seurat package
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| Submission date |
Jul 10, 2020 |
| Last update date |
Jul 09, 2023 |
| Contact name |
Stephanie Philtjens |
| E-mail(s) |
sphiltje@iu.edu
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| Phone |
3172786353
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| Organization name |
Indiana University School of Medicine
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| Street address |
320 W. 15th Street, NB Bldg Rm 103
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| City |
Indianapolis |
| State/province |
Indiana |
| ZIP/Postal code |
46202 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE154208 |
Chemogenetic activation of astrocytes in the hippocampus and cortex changes the transcriptome of microglia |
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| Relations |
| BioSample |
SAMN15504370 |
| SRA |
SRX8707787 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4666933_107_barcodes.tsv.gz |
60.4 Kb |
(ftp)(http) |
TSV |
| GSM4666933_107_features.tsv.gz |
272.8 Kb |
(ftp)(http) |
TSV |
| GSM4666933_107_matrix.mtx.gz |
42.3 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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