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| Status |
Public on Jun 20, 2020 |
| Title |
E3Ki_E4Mixed (scRNA-seq) |
| Sample type |
SRA |
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| Source name |
neurons / brain cells
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell type: transplanted hIPSC-derived neurons and surrounding hippocampal brain cells
treatment: Transplanted into E3KI Mouse Brain
genotype: human cells: ApoE4; mouse cells: E3KI
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| Treatment protocol |
Neurons (D28–35; i.e., +1-+2 weeks maturation) were washed in 1X PBS which was then aspirated and replaced with warm Accutase (Millipore) for 15 minutes or until neurons dissociated with gentle tapping. Accutase was neutralized with N2B27 medium to bring total volume >30 mL and then cells were filtered through a 40 mm strainer (Fisher) to ensure a single cell suspension. Single cells were then centrifuged and resuspended to concentration of 1000 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 for transplantation. ApoE4-KI and apoE3-KI 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). The head was secured with earbars and a tooth bar in a stereotaxic alignment system (Kopf Instruments). Fur was removed from the scalp, which was then sterilized with alternating swabs of chlorhexidine and 70% ethanol. The scalp was opened and sterilized with 3% hydrogen peroxide. Cell suspensions (~1000 cells/nL) were loaded into ~60 mm tip diameter, 30° beveled glass micropipette needles (Nanoject, Drummon Scientific Company). Bilateral rostral and caudal stereotaxic sites were drilled with a 0.5 mm microburr (Foredom, Fine Science Tools), and the coordinates used for hilar transplantation were X = ±1.65, Y = 2.00, Z = 1.7 and X = ± 2.90,Y = 3.20, Z = 2.2, with Z measured from the surface of the brain (David Kopf Instruments). At each transplantation site, ~20nL (~20,000 cells) were injected and allowed to diffuse for 3 min. For recovery, mice were sutured with 6–0 monofilament nonabsorbent nylon sutures (Ethicon), administered analgesics ketophen (5 mg/kg subcutaneously) and buprenorphine (0.0375 mg/kg intraperitoneally), and monitored on a heating pad until ambulatory.
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| Growth protocol |
Mixed hiPSC-derived neuronal populations were derived based on methods developed previously from our lab (Wang et al., 2018). Three 10cm-dishes (Corning) of 85% confluent hiPSCs-were dissociated with a brief treatment of Accutase (Millipore). After hiPSCs were dissociated, Accutase was neutralized by addition of equal volume of N2B27 medium (50% DMEM/F12 (Gibco), 50% Neurobasal Medium (Gibco), 1% NEAA (Gibco), 1% Glutamax (Gibco), 0.5% Pen/Strep (Gibco), 1X N2 Supplement [ Stock 100X] (Gibco), 1X B27 Supplement [Stock 50X] (Gibco)). hiPSCs were centrifuged and re-suspended in N2B27 medium with the addition of 10 mM SB (Stemgent) and 0.25 mM LDN (Stemgent) as well as 10 mM Rock Inhibitor (Tocris) in a T-175 flask (Thermo Scientific). After 24 hours, hiPSCs formed small spheres (neurospheres) and were centrifuged and resuspended in SB and LDN only. This was repeated every 48-hours until 7 days post differentiation. On day 7, the neurospheres were plated down onto two 10cm dishes coated with growth factor reduced Matrigel (Corning) and allowed to form neuronal rosettes. During this time rosettes were sustained by N2B27 media alone and half of the media was replaced every 48-72 hours depending on confluency. On day 20, rosettes had formed immature neuronal populations and were dissociated for maturation. Rosettes were treated with Accutase for 10-15 minutes until they dissociated under gentle pipetting. Once dissociated, neuronal progenitors were filtered through a 40 mm strainer (Fischer) to ensure a single cell suspension. They were then plated into tissue culture plates that were first coated with Poly-L-Lysine (Sigma, 0.1 mg/mL) overnight at 37°C, washed 3X with DPBS (Gibco) and then coated with Laminin (Gibco, 6 mg/mL) overnight at 37°C. Neurons were plated in N2B27 medium supplemented with 10 mM DAPT (Tocris), 10 ng/mL BDNF (Peprotech), and 10 ng/mL GDNF (Peprotech) for one week, after which DAPT was removed and BDNF and GDNF were maintained.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Hippocampi were isolated from single hemispheres of mice at the time of euthanasia, immediately frozen in dry ice and stored at -80°C. For isolation of human nuclei, hippocampi were thawed and incubated in HEB (BrainBits LLC)(1mL/ hippocampi) and kept on ice. 2mL chilled lysis buffer was then added to the tissue and was homogenized with 21G needle. Tissue was lysed on ice for 15 minutes and swirled to mix. This was repeated 3 times during incubation. Lysed hippocampi were titrated 5-7 times with 1mL pipette. Lysate was then washed in 30mm MACS Smart Strainer to remove large clumps and then centrifuged at 500g for 5 minutes are 4°C. Supernatant was aspirated and pellet was washed with 1mL Nuclei Wash and Resuspension Buffer. Nuclei were centrifuged at 500g for 5 min at 4°C and supernatant was then aspirated. The nuclear pellet was resuspended in 500mL staining buffer (0.5% RNase-free BSA and 0.2 mg/mL RNasin Plus RNAse inhibitory in RNAse-free PBS) and incubated for 15 minutes on ice to allow for blocking of non-specific antibody binding. 100mL of samples were removed for negative control staining. After blocking, anti-human nuclear antigen antibody conjugated to phycoerythrin was added to the nuclei solution [1:100] and incubated at 4°C for 30 minutes rotating. After incubation, samples were washed by adding 500mL of staining buffer to each tube and inverting several times. Samples were then collected by centrifugation for 5 min at 400g at 4°C. Supernatant was aspirated, and nuclei were resuspended in 500mL staining buffer for FACS sorting, with DAPI (0.1 mg/mL) added directly before sorting on Aria FACS sorter (BD Biosciences).
cDNA libraries were prepared using the Chromium Single Cell 3′ Library and Gel Bead kit v3 (10x Genomics: 1000092) 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 |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
hIPSC-derived apoE4 neurons transplanted into the E3KI mouse hippocamps, allowed to mature for 7 months. Collected with surrounding hippocampal tissue.
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| Data processing |
aligned with STAR using Cellranger v.2.0.1
UMIs assigned to barcodes and counted using Cellranger v.2.0.1
filtered UMI count matrices was loaded into Seurat v.2.3.4
Data were filtered to include only protein coding genes. Cells were filtered to include only cells with 100–1000 genes detected, 100–2000 UMI, and <0.05% mitochondrial reads
gene expression matrices were then log-normalized with a scale of factor of 10,000
Genome_build: GRCh38-3.3.0
Supplementary_files_format_and_content: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz
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| Submission date |
Jun 19, 2020 |
| Last update date |
Jun 20, 2020 |
| Contact name |
Kelly Zalocusky |
| Organization name |
Gladstone Institutes
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| Street address |
1650 Owens Street, Gladstone Center for Translational Advancement
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL25526 |
| Series (1) |
| GSE152867 |
In Vivo Chimeric Alzheimer’s Disease Modeling of Apolipoprotein E4 Toxicity in Human Neurons |
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| Relations |
| BioSample |
SAMN15327321 |
| SRA |
SRX8585944 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4628212_E3Ki_E4_barcodes.tsv.gz |
13.3 Kb |
(ftp)(http) |
TSV |
| GSM4628212_E3Ki_E4_features.tsv.gz |
297.6 Kb |
(ftp)(http) |
TSV |
| GSM4628212_E3Ki_E4_matrix.mtx.gz |
3.0 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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