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| Status |
Public on Jul 28, 2021 |
| Title |
MEF_Dox_Day2 |
| Sample type |
SRA |
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| Source name |
E13.5
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| Organism |
Mus musculus |
| Characteristics |
tissue: E13.5 embryo
cell type: Myocytes/Myotubes derived from MEFs
treatment: overexpressing MyoD for 2 days
genotype: C57BL6J/129Sv
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| Growth protocol |
MEFs harboring Rosa-rtTA, Col1a1-tetO-MyoD and Pax7-nGFP were isolated from E13.5 embryos and were maintained in DMEM (GIBCO) containing 1X glutaMAX (GIBCO), 1X MEM non essential Amino Acid Solution (GIBCO), 100 U/ml penicillin, 100 μg/mL streptomycin (GIBCO), 0.1% β-mercaptoethanol (GIBCO) and 10% Fetal Bovine Serum (HyClone). For dedifferentiation into iMPCs, MEFs were maintained in knockout DMEM (GIBCO) containing 1X glutaMAX (GIBCO), 1X MEM non essential Amino Acid Solution (GIBCO), 100 U/ml penicillin, 100 μg/mL streptomycin (GIBCO), 0.1% β-mercaptoethanol (GIBCO), 10% KnockOut Serum Replacement (GIBCO), 10% Fetal Bovine Serum (HyClone), 10ng/ml basic FGF (Peprotech), 5μM Forskolin (Sigma), 5μM RepSox (Sigma), 3μM GSK3β inhibitor CHIR99021 (Tocris) and 2μg/ml Doxycycline (Sigma). For transdifferentiation into myotubes, MEFs were maintained in knockout DMEM (GIBCO) containing 1X glutaMAX (GIBCO), 1X MEM non essential Amino Acid Solution (GIBCO), 100 U/ml penicillin, 100 μg/mL streptomycin (GIBCO), 0.1% β-mercaptoethanol (GIBCO), 10% KnockOut Serum Replacement (GIBCO), 10% Fetal Bovine Serum (HyClone), 10ng/ml basic FGF (Peprotech) and 2μg/ml Doxycycline (Sigma). Hypoxia experiments were performed in 5% O2 level. Freshly isolated satellite cells (fSCs) were isolated from adult Pax7-nGFP mice (10-12 weeks). Cultured satellite cells (cSCs) were maintained in F10 (GIBCO) with 20% Horse Serum (GIBCO), 1X glutaMAX (GIBCO) and 100 U/ml penicillin, 100 μg/mL streptomycin (GIBCO). 5ng/ml basic FGF (Sigma) was daily added. For differentiating cSCs, cSCs were cultured in 2% horse serum differentiation medium for 2 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells (1000 cells/μl) were resuspended with PBS and were loaded into Chromium 10x system.
scRNA-seq libraries were generated from approximately 5000 cells per sample using Chromium 10x instrument with version 3.0 kit.
single cell RNA-sequencing
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
sequenced reads were aligned to the mm10 reference genome using CellRanger version 3.0.2.
The filtered reads assigned to cell barcodes were analyzed with the R package Seurat, version 4.0.0 (Hao, 2020). In an initial quality control step, all cells with less than 300 genes expressed were removed, as well as all genes expressed in less than 10 cells (in CreateSeuratObject function). In a second step, only cells with less than 20% percentage mitochondria expression were retained.
The data was normalized using SCTransform (Hafemeister and Satija, 2019) and decomposed using PCA.
UMAP (McInnes, 2020) embeddings were then computed using the first 30 dimensions as input. Based on the UMAP embedding, further quality control was performed by removing clusters of contaminating cells (identified by differential expression and relevant marker genes of contaminating populations) and of poor-quality cells (identified as outliers by number of genes expressed or percentage mitochondria).
Genome_build: mm10
Supplementary_files_format_and_content: text files containing SCT-normalized expression values (the 'data' slot in the Seurat object) for each analyzed cell after QC.
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| Submission date |
Mar 29, 2021 |
| Last update date |
Jul 28, 2021 |
| Contact name |
Simona Cristea |
| Organization name |
Dana-Farber Cancer Institute
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| Department |
Biostatistics and Computational Biology
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| Street address |
450 Brookline Ave.
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE171039 |
Dissecting mechanisms by which MyoD and small molecules convert fibroblasts to muscle progenitor cells |
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| Relations |
| BioSample |
SAMN18524921 |
| SRA |
SRX10466196 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5217009_preprocessed-MEF_Dox_Day2.txt.gz |
20.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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