 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 23, 2021 |
| Title |
MEF14_Dox+_Day4_rep1 [RNA-seq] |
| Sample type |
SRA |
| |
|
| Source name |
MEF14_Dox+_Day4
|
| Organism |
Mus musculus |
| Characteristics |
cell type: MEF14_Dox+_Day4
|
| Treatment protocol |
Cells were harvested following trypsin treatment and processed for RNA extraction.
|
| 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. Primary satellite cells were isolated from adult Pax7-nGFP mice (10-12 weeks). Expanded satellite cells 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.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using miRNeasy Mini Kit according to the manufacturer’s instructions.
RNA-seq libraries were constructed from polyadenosine (polyA)-selected RNA using NEBNext Ultra Directional RNA library prep kit for Illumina (New England BioLabs). Libraries were amplified for 14 cycles. Post library constructions, the samples were validated using 2200 Tapestation System and High Sensitivity D1000 ScreenTape kit. Libraries were quantified using the Kapa Biosystems Library Quantification kit (KK4828) and the BioRad CFX96 instrument. Each lane of sequencing was pooled into a 19-plex (19 samples per lane) with unique barcodes. Pooled libraries are also quantified using the Kapa Biosystems Library Quantification kit (KK4828) and the BioRad CFX96 instrument. These pools are then denatured to 16 pM with 1% PhiX and sequenced on the Illumina HiSeq2000 instrument, resulting in approximately 30 million reads per sample on average.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to reference genome mm9 using STAR.
Transcript abundance was calculated using Htseq.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: *.count.txt: Tab-delimited text files include raw count for each gene.
|
| |
|
| Submission date |
Mar 23, 2021 |
| Last update date |
Aug 24, 2021 |
| Contact name |
Fei Ji |
| Organization name |
Massachusetts General Hospital
|
| Street address |
185 Cambridge St
|
| City |
Boston |
| ZIP/Postal code |
02129 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE169487 |
Induced muscle progenitor cells [RNA-seq] |
| GSE169489 |
Induced muscle progenitor cells |
|
| Relations |
| BioSample |
SAMN18448425 |
| SRA |
SRX10430850 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5206996_MEF14_Dox+_Day4_rep1.count.txt.gz |
140.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
