|
| Status |
Public on Sep 26, 2019 |
| Title |
enNOS-60-mpw-B |
| Sample type |
SRA |
| |
|
| Source name |
Wounded tissue collected 60 minutes post wounding in embryos injected with morpholinos against nos1 and nos3
|
| Organism |
Xenopus laevis |
| Characteristics |
time after injury: 60 min
biological replicate: B
treatment: nos1 + nos3 morpholino
tissue: embryo
|
| Treatment protocol |
We removed the vitelline membrane one hour before wounding and incubated embryos in 0.1xMBS with gentamicin and TRIM (1-(2-Trifluoromethylphenyl) imidazole, Sigma). TRIM solution was prepared as 1 M stock solution in DMSO and 2 µL per mL were used in final solution.
A 2:1 mix of morpholino-oligonucleotides targeting against nos3 (eNOS) and nos1 (nNOS) was prepared at a final concentration of 17 ng/µL. Two nanolitres of this mix was then injected into the fertilized oocytes.
|
| Growth protocol |
Embryos were grown at 15 °C for 3 days up to the NF stage 26 in 0.1xMBS with addition of gentamicin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using TRIreagent (Sigma) following the manufacturer's protocol. Diluted RNA was treated with DNAse I solution (Sigma), followed by an overnight reprecipitation during incubation at -20 °C using a 4 M final concentration of LiCl solution .
RNA-Seq was performed using biological duplicates. Kit from NewEnglandBiolabs (NEB #E7490S) was used for poly-A selection of RNA using 500 ng of total RNA. Libraries were prepared by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB #E7420S) according to manufacturer's protocol and sequenced on the NextSeq 550 in 2x75bp HighOutput mode.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
Low quality reads and adaptor sequences were removed using TrimmomaticPE (v. 0.36). Ribosomal and mitochondrial RNA sequences were removed using sortmerna (version 2) and the SILVA rRNA database (version 119). Reads were aligned against the Xenbase Xenopus genome version 9.1 using STAR (version 2.5.2b). Count tables were generated to annotation version 1.8.3.2 using htseq-count (v. 0.6.1p1).
Normalized number of reads was generated by DESeq2 using default parameters.
Genome_build: 9.1
Supplementary_files_format_and_content: Tab-delimited text files include non-normalized and DESeq2 normalized number of reads for each Sample
|
| |
|
| Submission date |
Jul 05, 2018 |
| Last update date |
Sep 26, 2019 |
| Contact name |
Radek Šindelka |
| Organization name |
Institute of Biotechnology, Czech Academy of Science, v.v.i.
|
| Lab |
Laboratory of Gene Expression
|
| Street address |
Průmyslová 595
|
| City |
Vestec by Prague |
| ZIP/Postal code |
25250 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL25291 |
| Series (1) |
| GSE116678 |
Gene expression during embryonic wound healing of Xenopus laevis after inhibition of production of NO. |
|
| Relations |
| BioSample |
SAMN09606931 |
| SRA |
SRX4343715 |
