|
| Status |
Public on Jun 30, 2024 |
| Title |
AJ11: RBP A C2 NC2 VSMC LOW |
| Sample type |
SRA |
| |
|
| Source name |
hES-VSMCs
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: Treatment
gfp intensity: Low
|
| Treatment protocol |
VSMCs were grown either untreated (NO dox) or treated with 0.2ug/ml Doxycycline for 10-14 days post differentiation in media containing 10% FBS.
|
| Growth protocol |
Human Embryonic stem cell derived Vascular Smooth Muscle Cells (neural crest lineage) were cultured in DMEM media supplemented with 10% FBS, Pen/Strep and L-GLutamine for 10-14 days post differentiation in 10cm dishes (BD Falcon) coated with 0.1% Gelatin in PBS. Protocols for differentiation from hES to Neural crest to VSMCs - refer Granata et al, 2017 (PMID 27893734).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by trypsinization and resuspended in FACS buffer (1% FBS in PBS supplemented with 2mM EDTA. Cells were sorted (BD FACSAria III) for Low, medium and High intensity GFP and collected in FACS Buffer. Sorted cells were spun down and resuspended in RLT buffer (RNAeasy, Qiagen) and frozen. All samples were extracted simultaneously using the RNeasy kit and resuspended in RNAse free water.
NovaSeq, PolyA selection beforehand (NEBNext® Poly(A) mRNA Magnetic Isolation Module, E7490), NEBNext® Ultra8482 II Directional RNA Library Prep Kit for Illumina (E7760)
bulk mRNAseq, paired-end, 150nt
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Rep_1
|
| Data processing |
Quality checks were performed using FastQC (v0.11.2), summarised with MultiQC (v1.8). Adapter trimming was performed using TrimGalore (v0.6.4_dev) and alignment to the human reference genome (build GRCh38.p13) was performed using STAR (STAR_2.5.2a) with default parameters. Density plots, violin plots, MA plots, Jaccard similarity index, dendrograms and PCA were performed as further quality checks
The data was normalised using quantile normalisation and noise was removed using a fixed threshold of 100 (only genes entirely under the threshold were discarded and abundances <100 were set to 100). An alternative noise removal method based on [Mohorianu et al, 2017; https://doi.org/10.1371/journal.pone.0182694] was also implemented. Differential expression analysiss was performed using edgeR (v3.28.1).
Enrichment analysis on the set of differentially expressed genes against a background of all genes expressed in the dataset using the g:profiler (https://biit.cs.ut.ee/gprofiler/gost) R package (v0.1.8).
Alternative splicing analysis was performed using rMATS [Shen et al, 2014; https://doi.org/10.1073/pnas.1419161111]
Genome_build: GRCh38.p13
Supplementary_files_format_and_content: Csv of raw and normalised abundances. Rows represent features and columns represent samples. Values are raw in the first instance, and normalised with noise removed in the second.
|
| |
|
| Submission date |
Jul 01, 2020 |
| Last update date |
Jun 30, 2024 |
| Contact name |
Irina Mohorianu |
| E-mail(s) |
data-submissions@stemcells.cam.ac.uk
|
| Organization name |
University of Cambridge
|
| Department |
Wellcome-MRC Cambridge Stem Cell Institute
|
| Street address |
Puddicombe Way
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0AW |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE153614 |
Splicing regulatory network of master regulator RNA Binding Protein Multiple Splicing (RBPMS) in human Embryonic stem cell derived Vascular Smooth Muscle Cells. |
|
| Relations |
| BioSample |
SAMN15415039 |
| SRA |
SRX8646445 |
