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| Status |
Public on Apr 05, 2020 |
| Title |
EC5 |
| Sample type |
SRA |
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| Source name |
Frozen post-mortem human brain tissue
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| Organism |
Homo sapiens |
| Characteristics |
brain region: Entorhinal cortex
batch: B
donor id: 5
braak stage (ad neuropathological progression): 2
Sex: Male
age (years): 77
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| Extracted molecule |
total RNA |
| Extraction protocol |
Isolation of nuclei was performed similarly as described in Mo et al. [PMID: 26087164]. Briefly, frozen brain tissue was dounce homogenized in 5 ml of lysis buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, pH 7.8, 1 mM DTT, 0.15mM spermine, 0.5 mM spermidine, 1X protease inhibitor (Sigma, 4693159001), and RNAse Inhibitor (Promega, N2615)). Following initial dounce homogenization, IGEPAL-630 was added to a final concentration of 0.3% and the sample was homogenized with 5 more strokes. The solution was then filtered through a 40 um cell filter and mixed with Optiprep (Sigma, D1556-250ML) to create a 25% Optiprep solution. This solution was then layered onto a 30%/40% Optiprep gradient and centrifuged at 10,000g for 18 minutes using the SW41-Ti rotor. The nuclei were collected at the 30%/40% Optiprep interface.
Droplet-based single-nuclues RNA-sequencing (snRNA-seq) was performed using the Chromium Single Cell 3′ Reagent Kits v2 from 10X Genomics. Nuclei were resuspended to a concentration of 1000 nuclei/uL in 30% Optiprep solution before loading according to manufacturer’s protocol, with 10,000 nuclei recovered per sample as the target. cDNA fragment analysis was performed using the Agilent 4200 TapeStation System. Sequencing parameters and quality control were performed as described by The Tabula Muris Consortium [PMID 30283141].
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Sequencing data generated from snRNA-seq libraries were demultiplexed using cellranger mkfastq.
To align reads, we first generated our own pre-mRNA GRCh38 reference genome using cellranger mkref in order to account for introns that may be eliminated using the default GRCh38 reference genome.
Alignment and gene expression quantification was then performed using cellranger count with default settings.
Genome_build: GRCh38 pre-mRNA
Supplementary_files_format_and_content: The processed data files are hdf5 formatted matrices of UMI counts, with rows as genes and columns as cell barcodes, obtained from running cellranger count.
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| Submission date |
Mar 25, 2020 |
| Last update date |
Aug 05, 2020 |
| Contact name |
Kun Leng |
| E-mail(s) |
kun.leng@ucsf.edu
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| Organization name |
University of California San Francisco
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| Department |
Institute for Neurodegenerative Diseases
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| Street address |
675 Nelson Rising Lane, 3rd Floor
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| City |
San Francisco |
| State/province |
Calfironia |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE147528 |
Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease |
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| Relations |
| BioSample |
SAMN14448159 |
| SRA |
SRX8001152 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4432651_EC5_raw_gene_bc_matrices_h5.h5 |
12.2 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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