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Status |
Public on Apr 07, 2024 |
Title |
CTX CTRL |
Sample type |
SRA |
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Source name |
corrected AxD (GFAP (R239C)
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Organism |
Homo sapiens |
Characteristics |
sample type: cortical organoids age: 165 days cell line: corrected AxD (GFAP (R239C) genotype: GFAP corrected
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Growth protocol |
Briefly, the culture of unguided organoids was started from single-cell suspensions of iPSCs (approx. 9000 cells/well) loaded into 96-well plates. At day 6 of cultivation, the medium was changed to neural induction medium. At day 13, the organoids were embedded in Matrigel and were further grown in culture dishes. At day 17, the organoids were transferred to orbital shaker. The medium was regularly changed and its components differed according to respective phases of cultivation. This protocol was inspired by Ormel et al. 2018 and Lancaster et al. 2013. The cortical organoids were cultivated according to the protocol of Yoon et al. 2019. In this case, the cells (approx. 3.5 x 106) were seeded into AggrewellTM800 plates. The embryoid bodies were tranferred to 96-well plates at day 2. From day 2 to day 6, the medium contained two SMAD inhibitors to allow for cortical specification.
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Extracted molecule |
polyA RNA |
Extraction protocol |
Single-cell suspension was prepared from organoids using combination of mechanical and enzymatic dissociation. The organoids were chopped to small pieces, treated with papain, and repeatedly resuspended by pipetting and subsequently incubated on an orbital shaker. The single-cell suspension was then fixed using 10x Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling, 10x Genomics protocol and kit, and the sequencing libraries were prepared using Chromium Fixed RNA Kit. Chromium Fixed RNA Kit (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 preprocessed_data.txt processed_data_Ctx.txt metadata_Ctx.csv
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Data processing |
Reads containing gene sequences were trimmed to uniform length 50 bp by TrimmomaticSE (0.36). Quality control was performed using FastQ Screen (0.11.1). STARsolo (STAR 2.7.9a) was used for alignment to reference genome. EmptyDrops (DropletUtils R package 1.16.0) with a threshold of 1000 UMIs and FDR <= 0.01 was used to filter out empty droplets. R language (4.2.2) and Seurat package (4.3.0) were used for downstream analysis. Samples were SCTransformed and integrated, excluding mitochondrial genes prefixed by MT-. Doublets were identified and filtered out using DoubletFinder (2.0.3). Cells with percentage of mitochondrial reads > 15 % were filtered out. Unguided and cortical samples were further analyzed separately using Seurat. The unguided samples were normalized, scaled, SCTransformed, and clustered. Analysis of cortical samples included one extra integration step due to great difference of the control and AxD samples. Clusters in both datasets were annotated based on expression of marker genes. 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 the RNA assay, counts slot of the Seurat object. Genes in rows, individual cells named by specific barcode and sample name in columns. Comma separated. All samples are included in one file. Supplementary files format and content: processed_data.txt files contain normalized data from the RNA assay, data slot of the Seurat object. Unguided and cortical samples were analyzed separately, therefore, we provide two separate preprocessed_data files. This is the dataset after filtering and QC. 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 objects and contain additional information about individual cells including the transcript numbers, cluster and sample classification, or cell cycle phase score. Cell identificators are in rows, names of variables are in columns. Unguided (metadata_CER.csv) and cortical (metadata_Ctx.csv) metadata are provided in separate files.
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Submission date |
Mar 08, 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) |
GSE261157 |
Aberrant neurodevelopment in human iPS cell-derived neural organoid model of Alexander disease |
GSE261158 |
Aberrant neurodevelopment in human iPS cell-derived models of Alexander disease. |
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Relations |
BioSample |
SAMN40310739 |
SRA |
SRX23880861 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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