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| Status |
Public on Apr 28, 2023 |
| Title |
scRNA-seq of the whole wt stembryos at 96h, rep2 |
| Sample type |
SRA |
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| Source name |
stembryo
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| Organism |
Mus musculus |
| Characteristics |
tissue: stembryo
cell line: mES cells
genotype: wt
stage (in_hours_after_aggregation): 96
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| Extracted molecule |
total RNA |
| Extraction protocol |
48 to 96 gastruloids were collected 96h or 120h after aggregation and washed twice in 1ml of PBS. Gastruloids were dissociated in 100ul of Stempro Accutase© for 5 minutes at 37°C, full dissociation was ensured by mechanical dissociation (pipetting) and verified to ensure absence of doublet cells. Then gastruloid cells were washed twice in 500ul PBS, and resuspended in an adequate volume of PBS 0.04%BSA at 106 cells/ml. All centrifugation steps were done for 5 minutes at 350G in DNA low binding Eppendorf tubes. Single cell suspensions were filtered using Flowmi Cell Strainer 40um and then were subjected to single cell RNAseq using the 10x genomics platform (V3, and V3.1 chemistry) following manufacturer’s recommendation.
cDNA preparations were performed according to 10x Genomics recommendations and was amplified for 10 to 12 cycles. And sequenced on a Hiseq 4000 with the cbot2 chemistry. At least 2 replicates were performed for each time points. In addition, a third 120h timepoint was generated by dissection in two portions to separate the anterior and posterior halves and each portion was analyzed separately by single cell RNAseq.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
All scripts are available on https://github.com/lldelisle/scriptsForRekaikEtAl2022
The cellranger pipeline version 6.0 (10x Genomics) was used with the default parameters on each fastq pair of scRNAseq datasets to perform the alignment, applying filtering and count barcodes and UMIs. Alignment was performed against the mouse reference genome mm10 and a modified gtf file based on Ensembl version 98 (https://doi.org/10.5281/zenodo.4456701). Each gene-cell count matrix was then analyzed using Seurat version 4.0.2 (Stuart et al. 2019). A Seurat object was generated by filtering out barcodes with less than 200 identified gene and genes identified in less than 3 cells. Then a further filtering was applied to remove low quality cells as well as doublets. For each dataset, the mean RNA count was used and cells with either less than 40% of the mean? or more than 2.5-fold the mean were remove. In addition, cells with more than 8% or less than 0.05% mitochondrial counts were removed. Each dataset was then normalized using the NormalizeData command, the 3000 most variable features were identified, and cell cycle was scored (using the 2019 updated gene list from Seurat). Then, all individual datasets were merged into a single Seurat object. The merged dataset was then normalized, the 3000 most variable features were identified. Then data scaling was performed using the ScaleData command using the percentage of mitochondrial count and cell cycle score.
Assembly: mm10
Supplementary files format and content: combined.96h.120h.rds: final R object containing filtered cells with expressions, clustering, projection etc. savec with saveRDS
Supplementary files format and content: filtered_feature_bc_matrix.tar: tar archive with output of cellranger (raw counts, gene names and cell names)
Supplementary files format and content: metadata.csv.gz: comma separated table containing metadata for each cell (clustering, sample of origin etc)
Supplementary files format and content: raw_counts.csv.gz: comma separated table contaiing the raw counts (genes as row and cells as columns)
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| Submission date |
Feb 09, 2023 |
| Last update date |
Apr 28, 2023 |
| Contact name |
Lucille Lopez-Delisle |
| E-mail(s) |
lucille.delisle@epfl.ch
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| Organization name |
EPFL
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| Street address |
Station 19
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| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE205783 |
Sequential and directional insulation by conserved CTCF sites underlies the Hox timer in stembryos |
| GSE224905 |
Sequential and directional insulation by conserved CTCF sites underlies the Hox timer in stembryos [scRNA-seq] |
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| Relations |
| BioSample |
SAMN33220239 |
| SRA |
SRX19486505 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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