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Sample GSM8136543 Query DataSets for GSM8136543
Status Public on Apr 07, 2024
Title CTX CTRL
Sample type SRA
 
Source name corrected AxD (GFAP (R239C)
Organism Homo sapiens
Characteristics sample type: cortical organoids
age: 165 days
cell line: corrected AxD (GFAP (R239C)
genotype: GFAP corrected
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.
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)
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description Read1 contains cell barcode and UMI, Read2 covers the gene sequences
preprocessed_data.txt
processed_data_Ctx.txt
metadata_Ctx.csv
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.
 
Submission date Mar 08, 2024
Last update date Apr 07, 2024
Contact name Zuzana Matusova
E-mail(s) zuzana.matusova@ibt.cas.cz
Organization name Institute of Biotechnology of the Czech Academy of Sciences
Department Laboratory of Gene Expression
Street address Prumyslova 595
City Vestec
ZIP/Postal code 25250
Country Czech Republic
 
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.
Relations
BioSample SAMN40310739
SRA SRX23880861

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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