|
| Status |
Public on Oct 29, 2019 |
| Title |
iPSC myotube-SDDF-RNAseq-Exp4 |
| Sample type |
SRA |
| |
|
| Source name |
iPSC myotube-SDDF
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human iPSC derived myotubes
treatment: S/Da/De/F (SDDF)
time: 5-day treatment
|
| Treatment protocol |
Myotubes were generated upon a 5-day treatment with vehicle (DMSO) or small molecules (SDDF)
|
| Growth protocol |
Human iPSC were differentiated into myogenic progenitors using conditional expression of PAX7. Myogenic progenitors were induced to terminally differentiate in a serum free low nutrient medium supplemented with vehicle (DMSO) or small molecules (SDDF)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted in TRIzol reagent and column purified with DNAse treatment using the Purelink RNA mini kit as per manufacturer's instructions
RNA-seq: Dual-indexed libraries were generated using the TruSeq stranded mRNA library kit
ATAC-seq: 50,000cells from 2-day DMSO- and SDDF-treated cultures were washed with 200 µl of cold PBS then resuspended in 100 µl of cold lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630), spun at 500 g for 10 minutes at 4°C and resuspended in 50 µl of the transposition reaction mix. Transposition occurred at 37°C for 30 minutes, after which transposed DNA was purified using a Qiagen MinElute Kit and eluted in 10µl Elution Buffer. Final libraries were generated by primer extension of the transposed DNA followed by PCR amplification using Illumina-compatible adapter-barcodes.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
PLZ-MT-SDDF-Exp4_S6
|
| Data processing |
ATAC-seq analysis. Reads were aligned to the human genome (hg38) using Bowtie2 with the following parameters: -q -I 38 -X 2000 --local --dovetail --no-mixed --no-discordant. Aligned reads were filtered for PCR duplicates using Samtools and then processes with MACS 2.1 with the following parameters: --nomodel --nolambda --keep-dup all --call-summits -q 0.05 -B. Bigwig files for visualization on IGV were generated by converting the bedgraph files obtained from MACS2 using the Kent tool bedGraphToBigWig. To identify peaks unique to DMSO and SDDF-treated cells, we used the BEDTools package: summits from DMSO- or SDDF-treated cells were extended 50bp in both directions and then compared pairwise with BEDTools intersect (Rep1_vs_Rep2, Rep1_vs_Rep3, Rep2_vs_Rep3). Common peaks from the 3 comparisons (DMSO or SDDF) were then combined in a unique list and filtered using BEDTools merge. DMSO and SDDF peaks were then compared using BEDTools intersect using the -v function and a minimum overlap of 60% (-f 0.6 -F 0.6). Analysis of differential accessibility was performed by generating a list of peaks representative of all samples using the peak summits identified by MACS2. Each summit was extended 50bp in both directions and the resulting lists of peaks were combined, sorted and merged to obtain a dataset of unique and non-overlapping loci. This list was then used to extrapolate the sequencing depth coverage from each sample bedgraph file. Coverage files were then analyzed using DEseq2 to identify loci with differential chromatin accessibility.
RNA-seq analysis: 75bp FastQ paired-end reads (n=20.9 Million per sample) were trimmed using Trimmomatic (v 0.33). Quality control on raw sequence data was performed with FastQC. Reads were mapped to the human genome (hg38) reference using Hisat2 (v2.1.0). 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 CLC genomics work bench (Qiagen) 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: hg38
Supplementary_files_format_and_content: ATAC-seq: processed files contain the list of peak summits identified by MACS2 in each replicate
Supplementary_files_format_and_content: RNA-seq: processed files contain tab-delimited expression values following analysis using EdgeR
|
| |
|
| Submission date |
May 01, 2019 |
| Last update date |
Mar 30, 2023 |
| 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 |
GPL21697 |
| Series (1) |
| GSE130592 |
Combinatorial small molecule treatment enhances the in vitro maturation of pluripotent stem cell-derived myotubes |
|
| Relations |
| Reanalyzed by |
GSM7124225 |
| BioSample |
SAMN11553142 |
| SRA |
SRX5776098 |
