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Sample GSM6605856 Query DataSets for GSM6605856
Status Public on Mar 07, 2024
Title ABCP_3d
Sample type SRA
Source name induced pluripotent stem cells
Organism Homo sapiens
Characteristics cell line: FS13B
cell type: induced pluripotent stem cells
time point: 3 days
condition: ABCP
Treatment protocol For directed differentiation, the human iPSCs were plated as single cells at 4.0-5.0x10^4 cells/cm2 using Accutase and 10 µM Y27632. Two days in the maintenance media after the splitting, cells were supplied every 24 hours with the ABCP media (CDM-PVA media supplemented with 100 ng/ml Activin A, 10 ng/ml BMP4, 3 μM CHIR99021 and 1 µM PD0325901).
Growth protocol Human iPSCs (FS13B line) were cultured on plates coated with 10 µg/ml vitronectin in 37°C and 5% CO2. For maintenance, cells were daily supplied with E6 media supplemented with 2 ng/mL TGFb and 25 ng/mL FGF2, and were passaged every 5-7 days using 0.5 mM EDTA in PBS.
Extracted molecule total RNA
Extraction protocol Cells were washed once with PBS and treated with accutase for 5 minutes at 37°C for single cell dissociation.
RNA libraries were prepared for sequencing using standard Illumina protocols for 10X Single Cell GEX v3.
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Description Differentiating cells cultured in ABCP condition for 72h
Data processing Quality checks, UMI identification, alignment to the mouse reference genome and cell calling were performed using the cellranger software (v6.1.1) with default parameters. Violin plots of the distribution of counts, features, mitochondrial and ribosomal RNA were created and upper and lower bounds were introduced to avoid data heterogeneity and exclude potentially problematic cells.
The data was normalised using the Seurat R package (v4.0.5). Dimensionality reductions (PCA and UMAP), as well as clustering were also performed using the Seurat R package, with the number of clusters being the optimal selected by the software. For the subsequent analysis, we focused on the 3000 most variable genes. Markers for each cluster were identified by testing for differential gene expression, using the Seurat R package.
Clustering stability was evaluated using the ClustAssess R package ( Enrichment analysis on the markers identified per cluster was performed using the g:profiler ( R package (v0.2.1). RNA velocity trajectory analysis was performed with the velocyto software (v0.17.17) and the scvelo python package ( Further analysis was performed with the above tools, focused on sub-clusters of biological interest.
Assembly: Human reference, GRCh38 (GENCODE v32/Ensembl 98) Mouse reference, mm10 (GENCODE vM23/Ensembl 98)
Supplementary files format and content: Sparse matrix of raw abundances for each gene, per cell
Submission date Sep 29, 2022
Last update date Mar 07, 2024
Contact name Irina Mohorianu
Organization name University of Cambridge
Department Wellcome-MRC Cambridge Stem Cell Institute
Street address Puddicombe Way
City Cambridge
ZIP/Postal code CB2 0AW
Country United Kingdom
Platform ID GPL24676
Series (1)
GSE214443 Single cell RNA sequencing of human pluripotent stem cells directed toward the amnioblast lineage.
BioSample SAMN31092732
SRA SRX17748198

Supplementary data files not provided
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Raw data are available in SRA
Processed data are available on Series record

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