|
| Status |
Public on Feb 26, 2019 |
| Title |
RNAseq_Pax3_H9_1day_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
differentiating hES cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: differentiating hES cells
differentiation protocol: serum
time: day6 of differentiation
purification: GFP-sorted
doxycycline-treatment: 1-day
|
| Growth protocol |
Human ES cell differentiation was achieved by plating 10^6 cells in 6cm non-adherent Petri dishes and incubation at 37°C, 5% CO2 on a shaker at 60 RPM. After 2 days, mTeSR was replaced with EB Myogenic (EBM) Media supplemented with 10µM Y-27632 and 10µM GSK3 inhibitor (CHIR990217) [EB Myogenic media composition: IMDM supplemented with 15% FBS, 10% Horse Serum, 1% Penicillin/Streptomycin, 2mM Glutamax, 50 μg/ml Ascorbic acid, 4.5 mM Monothioglycerol]. 2 days later, media was replaced to remove GSK3 inhibitor and the formed EBs were plated on gelatin-coated flasks using EBM + 10ng/ml human basic FGF (bFGF). 24h later, cultures were supplemented with 1µg/ml doxycycline (dox) for PAX3 induction. 24h later cells were harvested using Trypsin+EDTA solution and FACS sorted based on GFP expression.
|
| 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 HiSeq 2500 |
| |
|
| Description |
processed data file:
RNAseq_human_iPAX3_1day.xlsx
RNAseq_H9_PAX3_1day_dox_03
|
| Data processing |
Human RNA-seq analysis: each sample’s reads were then processed using RSEM version 1.2.3 (with bowtie-0.12.9 for the alignment step) [103, 104]. Genes were filtered to select only those with median-normalized TPM (Transcripts Per Million mapped reads) greater than 10 in at least one sample, log2fold change greater than 1 and p<0.05.
Genome_build: hg38
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 |
GPL16791 |
| 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 |
SAMN10756540 |
| SRA |
SRX5255408 |
