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| Status |
Public on Mar 06, 2024 |
| Title |
scRNA_Embryo |
| Sample type |
SRA |
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| Source name |
cell line
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| Organism |
Mus musculus |
| Characteristics |
tissue: cell line
cell line: 3D epiblast stem cells
cell type: Mouse pluripotent stem cells
genotype: wild-type
treatment: Cultured in 3D Matrigel with FGF2-ActivinA-XAV-Noggin
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| Extracted molecule |
total RNA |
| Extraction protocol |
The single-cell suspension was washed three times in 0.4% BSA in 1X PBS by centrifugation at 4 °C for 5 minutes at 135 g in DNA Lobind tubes (0030108035, Eppendorf). The cells were counted with a hemocytometer (using Trypan blue staining), and the overall number was accounted for by counting dead cells as well. The number of input cells was determined following 10X genomics' recommendations for a sample recovery of 4,000 cells.
scRNAseq was performed on the cells using a 10X Genomics Chromium Single-cell 3' v2 kit. Except for the number of cycles used, single-cell libraries were generated according to the manufacturer's protocol. For cDNA amplification, 9 cycles in total were used. For library amplification, 8 cycles were used in total. The quality and concentration of cDNA and the library were assessed using a 4200 Agilent TapeStation device and D5000 ScreenTape reagents.
10X Genomic single-cell 3' RNA-seq
Accel-NGS Methyl-Seq DNA Library Kit (manufacturer's recommendation)
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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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| Description |
10x Genomics
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| Data processing |
10X Genomic single-cell 3' RNA-seq
Raw base call (BCL) files generated by Illumina sequencers were demultiplexed into FASTQ files using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("mkfastq" pipeline, default parameters).
For each experiment FASTQ files were aligned, filtered, barcode and UMI counted using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("count pipeline", default parameters).
Assembly: mm10
Supplementary files format and content: For all samples there is one tar gz archive containing the gene-barcode matrices as produced by the Cell Ranger pipeline. They contain the MEX-formatted UMI count matrices of each sample, or if multiple libraries where sequence, the aggregated gene-barcode matrix.
PolyA RNA-seq
All RNAseq samples were pre-processed using cutadapt to remove adapter and trim low quality bases.
Reads were subsequently aligned against the human reference genome mm10 using STAR (parameter: outSAMtype BAM SortedByCoordinate --outSAMattributes Standard --outSAMstrandField intronMotif --outSAMunmapped Within --quantMode GeneCounts).
Stringtie was used for calculation of strand-specific TPMs.
Assembly: mm10
Supplementary files format and content: tsv of TPM values
Total RNA-seq
All RNAseq samples were pre-processed using cutadapt to remove adapter and trim low quality bases.
To remove rRNA all reads were first aligned against the rDNA databank (rfam-5s-database-id98) using STAR and unmapped reads were kept for the next step.
Reads were subsequently aligned against the human reference genome mm10 using STAR (parameter: outSAMtype BAM SortedByCoordinate --outSAMattributes Standard --outSAMstrandField intronMotif --outSAMunmapped Within --quantMode GeneCounts).
Stringtie was used for calculation of strand-specific TPMs.
Assembly: mm10
Supplementary files format and content: tsv of TPM values
Bisulfite-Seq
Bisulfite-converted reads were quality filtered and adapter trimmed using cutadapt (v2.4), followed by alignment to the reference genome with bsmap using the following parameter: -v 0.01 -s 16 -w 100 -S 1 -R -u -q 20. Duplicate reads were removed using GATK MarkDuplicates (v4.1.0.0).
Methylation rates were called using mcall using default parameter and subsequently filtered for coverage (min 10 reads, max 150 reads).
Assembly: mm10
Supplementary files format and content: bigWig file of 10X coverage filtered methylation rate BED file (<chr> <start> <end> <methylation rate>)
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| Submission date |
Sep 14, 2022 |
| Last update date |
Mar 06, 2024 |
| Contact name |
Helene Kretzmer |
| E-mail(s) |
kretzmer@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Genome Regulation
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| Lab |
Meissner Lab
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| Street address |
Ihnestraße 63
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE213336 |
Basal cell extrusion primes pluripotent cells for differentiation |
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| Relations |
| BioSample |
SAMN30852890 |
| SRA |
SRX17578064 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6579114_scRNA_Embryo.tar.gz |
87.2 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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