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Sample GSM4975213 Query DataSets for GSM4975213
Status Public on Dec 14, 2023
Title 72h OE 2
Sample type SRA
 
Source name Oesophageal epithelial cells
Organism Mus musculus
Characteristics time point: 3 days
mesenchymal compartment re-epithelialised: Oesophageal stroma
replicate: Replicate_2
Growth protocol 3D organotypic cultures were performed by combining oesophageal epithelium with either oesophageal stroma or skin stroma (tail dermis) in ThinCert™ Cell Culture Inserts (6-well plates with a pore diameter of 0.4μm, Greiner Bio-One), in DMEM High Glucose/DMEM:F12 (1:1) supplemented with FBS 5%, Insulin 5ug/ml, Adenine 25ug/ml and Apotransferrin 10ug/ml.
Extracted molecule total RNA
Extraction protocol After 3 or 10days in culture, the oesophageal-derived epithelia were harvested and peeled off from the oesophageal stroma or dermis. The epithelial sheet was dissociated into a single cell suspension and FACS sorted to select live singlets. For each sample, 7,000-9,000 cells were submitted for 10X single cell GEX v3 library preparation.
RNA libraries were prepared for sequencing using standard Illumina protocols for 10X Single Cell GEX v3
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description oesophagus in vitro - 3 days
Data processing Quality checks, UMI identification, alignment to the mouse reference genome and cell calling were performed using the cellranger software (v3.1.0) 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 (v3.2.2). 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 1000 most abundant genes. Markers for each cluster were identified by testing for differential gene expression, using the Seurat R package. The above steps were performed both for all the cells in the data, but also individually for each sample set (HF_3d, HF_10d, OE_3d, OE_10d).
Clustering stability was evaluated by a PAC analysis (https://doi.org/10.1038/srep06207). Enrichment analysis on the markers identified per cluster was performed using the g:profiler (https://biit.cs.ut.ee/gprofiler/gost) R package (v0.1.8). RNA velocity trajectory analysis was performed with the velocyto software (v0.17.17) and associated R package (v0.6). Pseudotime analysis was performed with the monocle3 R package (v0.2.3.0). Further analysis was performed with the above tools, focused on sub-clusters of biological interest.
Cells marked with the fluorescent markers eGFP and tdTomato were identified through sequencing. The identity of the unlabelled cells was inferred by using a semi-supervised classifier based on random forests.
Genome_build: GRCm38.97
Supplementary_files_format_and_content: Sparse matrix of raw abundances for each gene, per cell
 
Submission date Dec 15, 2020
Last update date Dec 14, 2023
Contact name Irina Mohorianu
E-mail(s) data-submissions@stemcells.cam.ac.uk
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 GPL24247
Series (1)
GSE163218 Esophageal progenitor cell plasticity; a 3D organotypic model of epithelial regeneration
Relations
BioSample SAMN17085581
SRA SRX9686348

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

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