|
| Status |
Public on May 29, 2020 |
| Title |
W+S D7_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
D7 Wounded skin with EMSCSp treatment_replicate2
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
strain: NOD/SCID
cell type: human ES derived MSC
|
| Treatment protocol |
The excisional wound splinting model was generated as reported. For the EMSCSp group, around 40 spheres equivalent of 1×106 EMSC were directly dropped onto the surface of each wound right after the wounding. For the EMSCDiss group, EMSCDiss at 0.7×106 cells in 100 l PBS (70% of the total cell dose) per wound were evenly injected into the dermis in four spots around the wound, and 0.3×106 EMSCDiss (30% of the total cell dose) mixed with 30-l Matrigel were dropped onto the surface of the wound. For the vehicle control group, 100-l PBS alone per wound was evenly injected around the wound, and 30-l Matrigel was dropped onto the surface of the wound as above.
|
| Growth protocol |
EMSC were generated by inducing hESC to differentiate into MSC using the method we reported previously. EMSC were cultured in high-glucose Dulebcco’s modified Eagle’s medium supplemented with 20% fetal bovine serum at 37.0℃ in 5% CO2 incubator, and passaged every 5-7 days. MSC between passages 6-10 were used for all experiments. For spheroid formation, MSC were split and seeded on adhesion substrates by using handing drop method at a density of 2.5×104 cells/drop as we recently reported 15, and the MSC in the handing drop formed spheroids within 2-day culture. Either MSC spheroids or cells dissociated from MSC in monolayer were harvested for transplantation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were sacrificed at designated time points, skin samples including the wound and 2 mm of the surrounding skin were harvested using an 8-mm biopsy punch, and three wounds were pooled per group. The tissues were sliced into small pieces and homogenized with a pestle. Skin fragments were added into a lysis buffer provided by QIAGEN RNeasy kit to isolate total RNA according to the manufacturer’s instructions.
The integrity and quality of samples were verified using the Nano RNA Bioanalyzer Assay (Agilent Technologies). Samples with RNA integrity number larger than 9 were used for Next Generation Sequencing library preparation using the NEB Ultra Directional RNA Library Preparation Kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Mus musculus + Homo sapiens
|
| Data processing |
Paired-end sequencing was performed on the Illumina HiSeq2500 platform
We applied FastQC for a standard quality control of our 100-bp length, paired-end RNA-seq data generated by Illumina HiSeq2500, tested both the raw sequence data, and trimmed sequence data.
Trimmomatic was used to trim the adaptor, and discarded low quality reads and the reads with length less then 36 bp. After trimming, we reserved the paired reads and abandoned unpaired reads for next step analysis. Then cleaned reads (paired reads) were submitted to the tuxedo pipeline.
Tophat2 internally called bowtie2 to map raw reads against the human genome hg38 and mouse genome mm10 with 2 mismatches allowed
Cufflinks were applied to generate the Fragments Per Kilobase Million (FPKM) value for each gene, based on the hg38 or mm10 gene annotation GTF file of University of California Santa Cruz. Human and mouse genome sequences, annotation files, and genome indexes were downloaded from iGenome
Genome_build: human genome hg38 and mouse genome mm10
The RNA-seq data was statically analyzed with R. One-way ANOVA test and fold change were used to identify DEG. We downloaded all the gene sets used for function/pathway enrichment from Gene Set Enrichment Analysis of the Broad Institute, and elucidated the hypergeometric distribution to determine P values of each enriched pathway. The R package “gplots” and “ggpubr” were used to prepare all figures.
Supplementary_files_format_and_content: FPKM
|
| |
|
| Submission date |
Nov 07, 2018 |
| Last update date |
May 30, 2020 |
| Contact name |
Renhe Xu |
| E-mail(s) |
renhexu@um.edu.mo
|
| Organization name |
University of Macao
|
| Department |
Faculty of Health Sciences
|
| Lab |
Renhe Xu lab
|
| Street address |
Avenida de Universidade Taipa
|
| City |
Macao |
| ZIP/Postal code |
853 |
| Country |
China |
| |
|
| Platform ID |
GPL22245 |
| Series (1) |
| GSE122257 |
Topical application of hESC-derived mesenchymal stem cell spheres accelerates wound healing in a CXCL12-CXCR4 axis-dependent manner |
|
| Relations |
| BioSample |
SAMN10391265 |
| SRA |
SRX4990975 |
