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| Status |
Public on Jan 23, 2023 |
| Title |
C315_C6_Mouse_EB_3D_WT |
| Sample type |
SRA |
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| Source name |
Embryoid Bodies (EB)
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryoid Bodies (EB)
Stage: EB_3D
genotype: WT
genotype ext: WT
batch: 2
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| Growth protocol |
E14 mouse WT and mutant (Dnmt3a -/- and Dnmt3b -/-) ESCs were cultured in high-glucose DMEM (Euroclone) supplemented with 15% FBS (Millipore Corp., Billerica, MA, USA), 0.1 mmol/l nonessential amino acids (Invitrogen), 1 mmol/l sodium pyruvate (Invitrogen), 0.1 mmol/l β-mercaptoethanol, 1500 U/ml Leukemia Inhibitory Factor (LIF; Millipore), 25 U/ml penicillin, and 25 μg/ml streptomycin. To induce formation of EBs, Wild type and mutant ESCs were transferred using trypsin to low-attachment plates (CORNING) in Alpha-MEM (BE02-002F LONZA) supplemented with 10% KOSR (10828-028 GIBCO), 5% FBS (Millipore), 1% nonessential amino acids (Invitrogen), 1% sodium pyruvate (Invitrogen), 0.1 mmol/l β-mercaptoethanol, 25 U/ml penicillin and 25 μg/ml streptomycin. Medium was changed every 3 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Full length single cell RNA-seq was performed using a modified version of the Smart-seq2 protocol. Briefly, individual cells are sorted into 96 well plates containing lysis buffer in presence of RNase inhibitor, dNTPs and OligodT. Reverse transcription of the polyadenylated RNA will be performed with SuperScriptII and Template Switching Oligos. The resulting cDNA will be amplified with 25 cycles of PCR and libraries will be prepared for sequencing with standard NexteraXT Illumina protocol. Libraries were sequenced on Illumina NextSeq 500 System (single-end 75bp reads).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Following quality controls (performed with FastQC v0.11.2 ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc), sequencing reads were processed with Trim Galore! v0.5.0 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore) to perform quality and adapter trimming (parameters: --stringency 3 –q 20). Trimmed reads were next aligned to the mouse reference genome (mm10/GRCm38.p6) using STAR v2.7.1a 51 with options: --outFilterMultimapNmax 10 --outFilterMultimapScoreRange 1 –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.04. Gene expression levels were quantified with featureCounts v1.6.1 38 (options: -t exon -g gene_name) using the GENCODE Release M23 annotation. Multi-mapped reads were excluded from quantification.
Assembly: mm10/GRCm38
Supplementary files format and content: Tab-delimited text file of raw read counts matrix per gene/cell.
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| Submission date |
Jan 13, 2023 |
| Last update date |
Jan 23, 2023 |
| Contact name |
Andrea Lauria |
| E-mail(s) |
andrea.lauria@unito.it
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| Organization name |
University of Turin
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| Department |
Life Sciences and Systems Biology
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| Lab |
Functional Genomics, Epigenomics - S. Oliviero
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| Street address |
Via Accademia Albertina, 13
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| City |
Turin |
| ZIP/Postal code |
10123 |
| Country |
Italy |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE168415 |
DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells |
| GSE222844 |
DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells [scRNA-seq II] |
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| Relations |
| BioSample |
SAMN32729295 |
| SRA |
SRX19028004 |
| 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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