|
| Status |
Public on Nov 30, 2023 |
| Title |
60-mpa-B_regeneration |
| Sample type |
SRA |
| |
|
| Source name |
tail
|
| Organism |
Xenopus laevis |
| Characteristics |
tissue: tail
time: 60 minutes post amputation
treatment: amputated
|
| Treatment protocol |
Bulk RNA-Seq analysis was done to analyze the temporal changes in gene expression during the regeneration periods of 0, 0.5, 1.5, 3, 6 hpa and 1, 3, 7 dpa. The experiment was done in biological triplicates.
|
| Growth protocol |
Xenopus laevis females were stimulated with 500 U of human chorionic gonadotropin (Sigma-Aldrich). Eggs were collected the following day and fertilized by testes suspension which were surgically obtained from the male. After the removal of jelly coats by the 2% cysteine treatment, embryos were incubated in 0.1x MBS until the experimental procedure. Embryonic stages were determined based on Nieuwkoop and Faber table. Embryos were immediately transferred to the 0.1x MMR solution with gentamycin. Solution was changed every day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All samples were stored at -80°C. Total RNA was extracted from 20 embryos per sample at the regenerative stage. Total RNA was extracted using TriReagent extraction and LiCl precipitation (Sigma) according to the manufacturer’s instructions. The concentration of total RNA was determined using a spectrophotometer (Nanodrop 2000; ThermoFisher Scientific), and the quality of RNA was assessed using a Fragment Analyzer (Agilent, Standard Sensitivity RNA analysis kit, DNF-471).
100 ng of total RNA was used for library preparation using the Lexogen SENSE Total RNA-Seq Library Prep Kit.
Library quality was tested by capillary electrophoresis on Fragment Analyzer (Agilent, NGS High Sensitivity kit DNF-474). The libraries were pooled and sequenced using Illumina NextSeq 500, 2x76 bp.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Reads were filtered for low quality reads and adaptor sequences, using TrimmomaticPE (v. 0.36), while ribosomal RNA reads were filtered out using Sortmerna (v. 2.1b) (default parameters).
The cleaned reads were then aligned using STAR (v. 2.7.9a) to the Xenopus laevis genome version 10.1 and annotation version XENLA_10.1_GCF.
A count table was then generated using the python script htseq-count (v. 0.6.1p1) with the parameter “–m union”. The counts were normalized and analyzed using DESeq2 (v. 1.32.0)[84] under R (v. 4.1.0).
Outlier samples were assessed by analyzing the PCA of the 500 most variable genes.
Assembly: Xenopus laevis genome version 10.1
Supplementary files format and content: 01_bulk_data_non_normalized.xlsx: non-normalized count data
Supplementary files format and content: 01_bulk_data_normalized.xlsx: median of ratios normalized data using DESeq2. Outlier samples were removed.
|
| |
|
| Submission date |
Oct 13, 2023 |
| Last update date |
Nov 30, 2023 |
| Contact name |
Radek Sindelka |
| E-mail(s) |
Radek.Sindelka@ibt.cas.cz
|
| 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 |
GPL21248 |
| Series (2) |
| GSE245317 |
Characterization of regeneration initiating cells during Xenopus laevis tail regeneration [bulk RNA-Seq 1] |
| GSE245320 |
Characterization of regeneration initiating cells during Xenopus laevis tail regeneration |
|
| Relations |
| BioSample |
SAMN37810388 |
| SRA |
SRX22090406 |
