|
| Status |
Public on Feb 26, 2019 |
| Title |
RNAseq_Pax3_1day_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
differentiating mES cells
|
| Organism |
Mus musculus |
| Characteristics |
tissue: differentiating mES cells
differentiation protocol: serum
time: day4 of differentiation
purification: PDGFRα+FLK1- sorted
doxycycline-treatment: 1-day
|
| Growth protocol |
Mouse ES cell differentiation was achieved by diluting 40000 cells/ml in EB differentiation medium and incubation in an orbital shaker at 80 RPM. EB differentiation medium: IMDM supplemented with 15% FBS , 1% Penicillin/Streptomycin, 2mM Glutamax, 50 μg/ml Ascorbic acid, 4.5 mM Monothioglycerol. For Serum-free differentiation, FBS was replaced with an equivalent amount of Knock-outTM Serum Replacement (Invitrogen). Trangene induction was achieved by adding doxycycline (Sigma-Aldrich) to day 3 EBs cultures (final concentration 1µg/ml), and then maintained throughout the differentiation protocol by replacing the media (including dox) every two days. At day 5, EBs were disgregated and single cells were incubated for 20 minutes with PDGFRα-PE and FLK1-APC conjugated antibodies. PDGFRα+FLK1- cells were sorted using FACSAriaII (BD biosciences) and replated on gelatin coated dishes using serum- or serum-free EB differentiation media supplemented with 1µg/ml doxycycline and 10ng/ml mouse basic-FGF.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA-seq: sorted cells from murine and human differentiating ES cells were resuspended in Trizol. RNAs were extracted using the PureLink RNA mini prep kit following manufacturer's instruction (including in-column Dnase digestion).
RNA-seq libraries were generated using the TrueSeq stranded kit and dual-index adapter-barcodes following manufacturer's instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
processed data file:
RNAseq_mouse_iPax3_1day_6day.xlsx
RNAseq_Pax3_1day_plusdox_03
|
| Data processing |
Mouse RNA-seq analysis: 150bp FastQ paired-end reads were trimmed using Trimmomatic (v 0.33) enabled with the optional “-q” option; 3bp sliding-window trimming from 3’ end requiring minimum Q30. Quality control on raw sequence data for each sample were performed with FastQC. Read mapping was performed via Hisat2 (v2.0.2) using the mouse genome (mm10) as reference. Gene quantification was done via Cuffquant for FPKM values and Feature Counts for raw read counts. Differentially expressed genes were identified using the edgeR (negative binomial) feature in CLCGWB (Qiagen, Valencia, CA) using raw read counts. We filtered the generated list based on a minimum 2X Absolute Fold Change and FDR corrected p < 0.05.
Genome_build: mm10
Supplementary_files_format_and_content: RNA-seq: processed files contain tab-delimited expression values following analysis using EdgeR (mouse RNA-seq) and RSEM (human RNA-seq)
|
| |
|
| Submission date |
Jan 16, 2019 |
| Last update date |
Feb 26, 2019 |
| Contact name |
Alessandro Magli |
| E-mail(s) |
alemagli@gmail.com
|
| Organization name |
University of Minnesota
|
| Department |
Medicine
|
| Street address |
2231 6th St SE
|
| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE125203 |
Chromatin accessibility landscape upon induction of Msgn1, Pax3 and Myf5 in mesodermal cells and identification of conserved Pax3 binding sites and target genes during skeletal myogenesis |
|
| Relations |
| BioSample |
SAMN10756581 |
| SRA |
SRX5255399 |
