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| Status |
Public on Jun 26, 2024 |
| Title |
DE_dTAG_at_DE pool |
| Sample type |
SRA |
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| Source name |
7H OTX2-dTAG-EGFP
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| Organism |
Mus musculus |
| Characteristics |
cell line: 7H OTX2-dTAG-EGFP
cell type: Definitive Endoderm
strain: BL6/129 hybrid
treatment: DMSO_dTAG at 24hrs DE
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| Treatment protocol |
dTAG13 or DMSO vehicle added at primitive streak (PS) or 24hrs DE stage to deplete OTX2
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| Growth protocol |
All cells cultured at 37C, 5% CO2. EpiSCs are grown in N2B27, 20ng/ml Activin A, 12.5ng/ml FGF2, 175nM NVP. DE are differentiated in CDM, 40ng/ml Activin A, 3uM CHIR for 16hrs followed by 100ng/ml Activin A, 100nM LDN for 48hrs
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells at indicated stages were dissociated using Accutase to single-cell suspension inPBS-/- with 0.04% BSA. Samples were labeled with Cell Multiplexing Oligos (10x GenomicsChromium Next GEM Single Cell 3สน Reagent Kit v3.1 Dual Index) and live cells sorted using DAPI on a BD Influx Cell Sorter.
Two pools were created for multiplexing inPBS-/- with 0.04% BSA (targeting 10,000 cells/sample)- EpiSCs, DE with DMSO or dTAG13 added at PS stage; and DE with DMSO or dTAG13 added at 24hrs DE differentiation. Single-cell libraries were generated according to the manufacturer's instructions (10x GenomicsChromium Single Cell Gene Expression - Cell Multiplexing).
scRNA-seq (10x Genomics, multiplexed pooling)
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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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| Data processing |
Fastq files were processed using CellRanger (10x genomics cloud).
Raw h5 files were processed using CellBender to eliminate background reads, empty beads, and other artifacts (Fleming, et al., 2022). Scanpy (Wolf, et al., 2018) was used for downstream analyses. Cell clusters with high percentage of mitochondrial reads or a relatively low number of reads were eliminated.
Palantir (Setty, et al., 2019) was used for pseudo-time analyses
For differential expression analyses, Wilcoxon testing was performed and only genes with padj<0.05 and log2 fold-change>1 were considered as differentially expressed genes (DEGs). The scores/ranking per gene given by the scanpy.get.rank_genes_groups_df function were used.
Assembly: mm10
Supplementary files format and content: h5ad, txt
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| Submission date |
Jan 29, 2024 |
| Last update date |
Jun 26, 2024 |
| Contact name |
Ly-sha Ee |
| E-mail(s) |
lye4001@med.cornell.edu, worms917@yahoo.com
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| Organization name |
Weill Cornell Medicine
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| Street address |
413 E 69th Street
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| City |
New York |
| ZIP/Postal code |
100021 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE254428 |
Transcriptional remodeling by OTX2 directs specification and patterning of mammalian definitive endoderm [scRNA-seq] |
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| Relations |
| BioSample |
SAMN39650690 |
| SRA |
SRX23424258 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8042146_dTAG_at_DE1_2nddataset.h5ad.gz |
339.2 Mb |
(ftp)(http) |
H5AD |
SRA Run Selector |
| Raw data are available in SRA |
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