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| Status |
Public on Nov 27, 2024 |
| Title |
Rep4, HTO |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: brain
cell type: microglia
library type: HTO
genotype: GFP-M::Cx3cr1-CreERT2fl/fl::Rosa26-tdTomatotg/tg
Sex: female
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| Treatment protocol |
Mice were fed with BLZ-945 (MedChemExpress, HY-12768) at a dose of 200 mg/kg b.w. daily in 100 µl PB (Peanut butter, GoOn, Santé); controls received orally PB. BLZ-945 was delivered for 14 or 21 days to establish the kinetics of depletion; repopulation was allowed for 7 days without any treatment.
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| Extracted molecule |
protein |
| Extraction protocol |
Mice were sacrificed and subsequently brains were collected, placed in cold HBSS (without Ca2+ and Mg2+), and transferred into 30 % sucrose (w/v) in PBS for 48 h and frozen in Tissue Freezing Medium (Leica) at -80°C. The tissue was minced, transferred onto c-tubes containing 1,950 µl DMEM, and supplemented with 50 µl of Deoxyribonuclease I (Sigma Aldrich). Dissociation was performed with the MACS dissociator with heaters (Militenyi Biotec) for 22 minutes and later the samples were passed through 70 nm and 40 nm strainers into 50 ml cold HBSS with ions to stop the enzymatic reaction (EasyStrainer™, BioOne).
For flow cytometry, cells were suspended in the Stain Buffer (BD Pharmingen) with anti-mouse CD16/CD32 Fc Block™ 1:200 (BD Pharmigen) for 10 min. Next, anti-mouse CD11b antibody 1:250 (M1/70, BD Pharmigen) was added and cells were incubated for 20 min at 4 °C, then washed with Stain Buffer. Cells were sorted using purity precision mode on FACS Aria (BD FACSAria Cell sorter BD-Biosciences), into 200 μL of sterile PBS.
Preparation of gel beads in emulsion andlibraries were performed with Chromium Controller and Single-Cell Gene Expression v2 Chemistry (10X Genomics). Libraries’ quality and quantity were verified with a High-Sensitivity DNA Kit (Agilent Technologies, USA) on a 2100 Bioanalyzer (Agilent Technologies, USA).
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| Library strategy |
RNA-Seq |
| Library source |
other |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Hashtag-derived oligonucleotide (HTO)
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| Data processing |
The demultiplexing, barcode processing, gene counting and aggregation were made using the Cell Ranger software v3.0.1. (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Jul 05, 2024 |
| Last update date |
Nov 27, 2024 |
| Contact name |
Patrycja Rosa |
| E-mail(s) |
[email protected]
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| Organization name |
Nencki Institute of Experimental Biology, Polish Academy of Sciences
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| Lab |
Laboratory of Molecular Neurobiology
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| Street address |
Ludwika Pasteura 3
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| City |
Warsaw |
| ZIP/Postal code |
02-093 |
| Country |
Poland |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE271560 |
Tracking changes in functionality and morphology of repopulated microglia in young and old mice |
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| Relations |
| BioSample |
SAMN42337238 |
| SRA |
SRX25569407 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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