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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 07, 2024 |
| Title |
OGD astroC/neuroC |
| Sample type |
SRA |
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| Source name |
corrected AxD (GFAP(R239C))
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| Organism |
Homo sapiens |
| Characteristics |
cell line: corrected AxD (GFAP(R239C))
cell type: neurons and astrocytes derived from iPSCs
genotype: corrected neurons + corrected astrocytes
treatment: oxygen-glucose deprivation challenge
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| Treatment protocol |
For OGD (oxygen-glucose deprivation) challenge, the co-cultures were cultured in deoxygenated ischemic media, in 1 % O2, which was followed by a recovery period of 16 h in fresh media.
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| Growth protocol |
iPSCs were dissociated with accutase and plated on Matrigel-coated 6-well plates with mTeSR medium with ROCK inhibitor. Astrocyte differentiation was induced with Sox9 and Nfib carried by lentiviral vectors. Neurons were induced similarly using Ngn2. For co-culture, accutase-dissociated cultures of astrocytes and neurons were plated together: 3.9×104 iAs + 1.11×105 iNs and were maintained for 42 days before experiments.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were dissociated by accutase with actinomycin D, resuspended with wide-bore pipette tips, and strained before centrifugation. The suspension was centrifuged and resuspended two times. Concentrations were normalized to target concentration, samples were strained and counted. Volumes of samples were adjusted to target concentration of 700-1200 cells/µL and target cell count of 10000 cells per sample.
Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (10x Genomics, Pleasanton, CA)
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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 |
Read1 contains cell barcode and UMI, Read2 covers the gene sequences
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| Data processing |
Quality control of the sequencing data was performed using FastQ Screen (0.11.1). STARsolo (STAR 2.7.9a) was used for alignment to reference genome.
EmptyDrops (DropletUtils 1.16.0) with threshold of 2000 UMIs and FDR <= 0.01 was used to filter out empty droplets.
R programming language (4.1.1) and Seurat package (4.1.0) were used for the downstream analysis. Data were SCTransformed, integrated (excluding mitochondrial and ribosomal genes), and series of quality control and filtering steps were applied, including filtering for nFeature_RNA > 1500 and nCount_RNA > 2500, identification of doublets (DoubletFinder 2.0.3), and removal of MT- (mitochondrial genes) and MALAT1-high clusters.
SoupX package (1.5.2) was used to identify contaminating transcripts and correct expression values according to the estimated contamination fraction.
Following Seurat analysis pipeline, three cell groups (astrocytes, neurons, and less differentiated precursors) were identified in normalized, scaled, SCTransformed, and clustered dataset. These groups were then analyzed separately along with additional filtering steps, to keep only the high-quality data. Finally, they were merged again together into one Seurat object.
Assembly: Homo sapiens GRCh38
Supplementary files format and content: preprocessed_data.txt contains count matrix generated after initial integration step. Only EmptyDrops filtering was applied. These data are not normalized and were extracted from RNA assay, counts slot of the Seurat object. Genes in rows, individual cells named by specific barcode and sample name in columns. Comma separated.
Supplementary files format and content: processed_data.txt file contains normalized data from RNA assay, data slot of the Seurat object. This is the dataset after multiple rounds of filtering, QC, and merging of individually analyzed cell populations (iA, iN, precursors). Genes in rows, individual cells named by specific barcode and sample name in columns. Comma separated.
Supplementary files format and content: metadata.csv was extracted from the Seurat object with processed data and contains additional information about individual cells including the total transcript numbers, cluster and sample classification, or cell cycle phase score. Cell identificators are in rows, names of variables are in columns.
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| Submission date |
Mar 07, 2024 |
| Last update date |
Apr 07, 2024 |
| Contact name |
Zuzana Matusova |
| E-mail(s) |
zuzana.matusova@ibt.cas.cz
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| Organization name |
Institute of Biotechnology of the Czech Academy of Sciences
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| Department |
Laboratory of Gene Expression
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| Street address |
Prumyslova 595
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| City |
Vestec |
| ZIP/Postal code |
25250 |
| Country |
Czech Republic |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE261110 |
Aberrant neurodevelopment in human iPS cell-derived astrocyte-neuron co-culture model of Alexander disease |
| GSE261158 |
Aberrant neurodevelopment in human iPS cell-derived models of Alexander disease. |
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| Relations |
| BioSample |
SAMN40301301 |
| SRA |
SRX23874877 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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