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| Status |
Public on Oct 25, 2024 |
| Title |
E3KI |
| Sample type |
SRA |
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| Source name |
hippocampus
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| Organism |
Mus musculus |
| Characteristics |
tissue: hippocampus
cell type: Microglia
genotype: APOE3/3
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| Treatment protocol |
Human induced pluripotent stem cell (hiPSC)-derived neurons (at culture days 35-42) were collected and single cells were then centrifuged and resuspended to concentration of 500 cells/nL in 1X HBSS (GIBCO) supplemented with 10 ng/mL BDNF (Peprotech), 10 ng/mL GDNF (Peprotech) and 100 ng/mL DNaseI (Roche) and kept at 4°C until transplantation. Mice were anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (30 mg/kg) and maintained on 0.8%-1.0% isofluorane (Henry Schein). Mice were secured in a stereotaxic alignment system model 940 using earbars and a tooth bar (Kopf Instruments). The scalp was prepared by removing hair with scissors and sterilizing with 70% ethanol. The scalp was then cut open using a scalpel and sterilized with 70% ethanol. Cell suspensions (500 cells/nL) were loaded into 60 μm tip diameter, 30° beveled glass micropipette needles (Nanoject, Drummond Scientific Company). Approximately 30,000 cells per site were injected at 4 sites (2 per hemibrain) at a rate of 25 nL/sec and allowed to diffuse for 1 min. Each mouse received four total cell transplants, two per hemibrain hippocampus. Following surgery, mice were sutured with nylon monofilament non-absorbable 6-0 sutures (Henry Schein), and administered analgesics buprenorphine (0.0375 mg/kg intraperitoneally), ketofen (5 mg/kg subcutaneously), and saline (500µL intraperitoneally). Immunosuppressants were administered immediately after transplantation (day 0) via intraperitoneal injection followed by injections on day 2, 4, and 6 post transplantation.
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| Growth protocol |
APOE3 and APOE4 knock-in (E3KI and E4KI) mice on a C57BL/6 background were originally obtained from Taconic. All animals were bred in-house using trio breeding producing 8–10 pups per litter on average, which were weaned at 28 days. Male littermates at 3-5 months of age were randomly assigned to experimental groups. Animals were housed in a pathogen-free barrier facility on a 12 hr light cycle (lights on at 7 am and off at 7 pm) at 19–23°C and 30%–70% humidity. Mice were genotyped by polymerase chain reaction (PCR) of a tail clipping at weaning. All mouse experiments were conducted in accordance with the guidelines and regulations of the National Institutes of Health, the University of California, and the Gladstone Institutes under IACUC protocol AN117112.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The hippocampus of each mouse’s left hemi-brain was dissected on ice, rinsed with cold Dissection Buffer, and placed into a pre-chilled 12-well plate with 2 mL/well of Hibernate A Buffer. Hippocampi from 4-6 mice per condition were pooled into one well for further processing. Tissue from each pooled sample was moved to a 100mm dish, minced with a razor blade, then placed in a 5mL Eppendorf tube with 1mL pre-warmed Enzyme Buffer. Samples in Eppendorf tubes were then incubated in a 35°C water bath for 30 minutes, with gentle trituration at the 15-, 25-, and 30-minute timepoints. The homogenate was then filtered (70um MACS SmartStrainers) into a 50mL conical tube and centrifuged and washed at 4°C for a few times. The cells were resuspended in Blocking Buffer (BD Biosciences). After a 5-minute incubation at 4°C, cells were incubated in Antibody Solution (1X DPBS, 0.2% BSA, 1.89ug/mL CD45 PE-Cyanine7 (Thermofisher), 3.53ug/mL CD11b Brilliant Violet 421 (BioLegend)) for 40 minutes at 4°C. After being washed with 1X DPBS with 0.2% BSA and filtered through a 40um Flowmi Cell Strainer (Millipore Sigma) into a pre-chilled 5mL FACS tubes (Stem Cell Technologies), live single cells were identified and gated per standard sorting protocols, then further sorted to isolate a CD11b+/CD45int microglia population (Supp Fig 6) with a BD FACS Aria Fusion I or BD Aria II at 4°C with a 100um nozzle (20 psi). The sorted microglia were centrifuged at 500g at 4°C for 5 minutes, then loaded onto 10x Genomics Next GEM chip G at cell counts of approximately 25,000 cells.
The scRNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Library and Gel Bead kit v.3.1 (10x Genomics) according to the manufacturer’s instructions. Libraries were sequenced on an Illumina NovaSeq 6000 sequencer at the UCSF CAT Core.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
contains both cytoplasmic RNA and nuclear RNA
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| Data processing |
The demultiplexed fastq files for these samples were aligned to the standard mouse reference genome (2020 version, refdata-gex-mm10-2020-A) separately using the 10x Genomics Cell Ranger v7.0.0 count pipeline, as described in the Cell Ranger documentation. The include-introns flag for the count pipeline was set to true to count the reads mapping to intronic regions.
Assembly: The Homo sapiens APOE are genes of interest for this study. Custom mouse reference genome was made using these genes of interest , the reference mouse genome sequence (GRCm38) from Ensembl (release 98) and the mouse gene annotation file from GENCODE (release M23), similar to those used in 10x Genomics Cell Ranger mouse reference package mm10 2020-A.
Supplementary files format and content: Feature-barcode matrix files (zipped) in the Market Exchange Format (MEX Format) (https://www.10xgenomics.com/support/software/cell-ranger/analysis/outputs/cr-outputs-mex-matrices)
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| Submission date |
Nov 16, 2023 |
| Last update date |
Oct 25, 2024 |
| Contact name |
Nuo Chen |
| E-mail(s) |
norman.chen@gladstone.ucsf.edu
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| Organization name |
Gladstone Institutes
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| Department |
GIND
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| Lab |
Huang Lab
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| Street address |
1650 Owens St
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| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE248020 |
Microglia Depletion Reduces Human Neuronal APOE4-Driven Pathologies in a Chimeric Alzheimer’s Disease Model |
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| Relations |
| BioSample |
SAMN38286456 |
| SRA |
SRX22551016 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7904794_E3KI_filtered_feature_bc_matrix.tar.gz |
60.6 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
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