|
| Status |
Public on Jul 01, 2020 |
| Title |
Q133H differentiation T=48 rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Emerin-null H2K myogenic precursor cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: H2K myogenic precursor cells
|
| Growth protocol |
Proliferating myogenic progenitors were cultured in plates coated with gelatin at 37°C and 10% CO2 in DMEM containing 20% HI-FBS, 2% L-glutamine, 2% Chick embryo extract , 1% Pen/Strep and 20 units/ml γ-interferon . Differentiating cells were cultured in plates coated with gelatin in proliferative conditions for 24 h, then switched to differentiation media consisting of DMEM supplemented with 5% horse serum and 2% L-glutamine and grown at 37°C and 5% CO2. For EDMD mutant cell lines, puromycin was added to the media during proliferation and differentiation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the mRNeasy Plus Kit (Qiagen, Germantown, MD. USA, #74136) per manufacturer’s instructions. Three technical replicates per sample were performed for each biological replicate (n=2).
The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligoattached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and pair-end reads of 100 bp were read through on the DNBseq platform for the following data analysis study. For this step, the DNBseq platform combines the DNA nanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
BGISEQ-500 |
| |
|
| Description |
Q133H differentiation T=48
Emerin-null cells transduced with Q133H emerin mutant
Q133H_T24-VS-Q133H_T48.DEseq2_Method.GeneDiffExp.xls
WT_T48-VS-Q133H_T48.DEseq2_Method.GeneDiffExp.xls
|
| Data processing |
Purity of RNA was assessed using Nanodrop (ND-2000) and quantification was done using the Quant-IT RNA assay kit (ThermoFisher Scientific, #Q10213). BGI Genomics performed library preparation and sequencing using BGISEQ-500 platform.
BGI Genomics filtered reads using SOAPnuke and genome mapping was done to the mouse genome (mm10) using hierarchical indexing for spliced alignment of Transcripts-2 (HISAT2).
Differentially expressed genes was detected using DEseq2. Transcripts were considered to be significantly differentially expressed if the q-value <0.05. The q-value is a p-value corrected for the False Discovery Rate (FDR) and represents the standard in the field for determining significance.
All differentially expressed transcripts were also >= 2-fold increased or decreased.
KEGG pathway enrichment analysis was done using phyper, a function of R.
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| |
|
| Submission date |
Jun 10, 2020 |
| Last update date |
Jul 01, 2020 |
| Contact name |
Ashvin Iyer |
| E-mail(s) |
[email protected]
|
| Phone |
732-501-5702
|
| Organization name |
University of the Sciences
|
| Department |
Pharmaceutical Sciences
|
| Street address |
1325B Johnston Drive
|
| City |
Bethlehem |
| State/province |
Pennsylvania |
| ZIP/Postal code |
18017 |
| Country |
USA |
| |
|
| Platform ID |
GPL23479 |
| Series (1) |
| GSE152226 |
EDMD-causing emerin mutant myogenic progenitors exhibit impaired differentiation using similar mechanisms |
|
| Relations |
| BioSample |
SAMN15203211 |
| SRA |
SRX8524918 |
