|
| Status |
Public on Oct 20, 2022 |
| Title |
T4_biorep1 |
| Sample type |
SRA |
| |
|
| Source name |
Transitional gonad stage 4
|
| Organism |
Lates calcarifer |
| Characteristics |
tissue: Gonad
developmental stage: Adult
Sex: Intersex
|
| Growth protocol |
Asian seabass were reared in seawater conditions at a temperature range of 28-31 ℃
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy mini kit (Qiagen) and sRNA was purified using the mirVana miRNA isolation kit (Life Technologies). RNA concentration and integrity was measured on the NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific) and was visually assessed by agarose gel electrophoresis with ethidium bromide staining. RNA was stored at -80 ℃
sRNA cDNA libraries were constructed by ligating sRNA to 3' and 5' HD adapters. Ligated RNA products were reverse transcribed to cDNA and amplified by PCR. The cDNA products expected to contain 19-33 base pair inserts were purified by 8% polyacrylamide gel electrophoresis and ethanol precipitation
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
sRNA
|
| Data processing |
Obtain putative miRNA precursors: Map miRBase animal precursor sequence against the Asian Sea Bass genome using BLAST (E = 10e-6). Merge overlapping BLAST hits.
Validate putatitve miRNA precursor sequences: Align mature miRBase animal miRNAs against putative precursors using PatMaN allowing one mismatch only. Putatative miRNA precursors with a mature miRNA match were folded using RNAfold, and those forming a valid pre-miRNA hairpin structure were validated
Read trimming and filtering: HD adaptor sequences were trimmed by removing the first 4 bases of each read followed by the 3’ adaptor and preceding four bases. Sequences shorter than 18nt and comprised of two or fewer unique bases were removed from further analysis.
Novel miRNA prediction: Putative novel miRNA predictions were generated both miRCat and miRDeep2 algorithms using default parameters. Predictions were merged to obtain a non-redundant set of candidates and known miRNA families from miRBase identified previously were removed from the predicted novel miRNA set. Small RNA reads were aligned to predicted miRNAs and the read alignment pattern and secondary structure were checked manually to ensure that they are consistent with canonical miRNA biogenesis. Any predictions that did not meet these criteria were removed.
Determine raw read counts: Processed reads were aligned to annotated Asian seabass mature miRNAs using PatMan, generating a count for each miRNA isoform for a given annotated mature miRNA. PatMan output was further processed using a custom perl script in order to generate total counts for each annotate mature miRNA per sample.
Differential Expresson Analysis: DESeq2 (v 1.20.0) was used to identify differential expressed miRNAs between conditions from raw miRNA read count data. Default parameters were used, except for the ‘alpha’ parameter which was set at 0.05 when calling the DESeq2 'results' function.
Genome_build: ASM164080v1
Supplementary_files_format_and_content: Lates_calcarifer_gonads_sRNA-Seq_raw_counts.txt: A matrix of raw miRNA read counts for each sample, tab-separated values
Supplementary_files_format_and_content: Lates_calcarifer_mature_miRNAs.fa: Mature miRNA sequences in FASTA format
|
| |
|
| Submission date |
Nov 19, 2018 |
| Last update date |
Oct 20, 2022 |
| Contact name |
László Orbán |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Pannonia
|
| Department |
Department of Animal Sciences
|
| Street address |
Egyetem u. 10
|
| City |
Veszprém |
| ZIP/Postal code |
8200 |
| Country |
Hungary |
| |
|
| Platform ID |
GPL25830 |
| Series (1) |
| GSE122720 |
sRNA sequencing of mature and developing gonads during protandrous changes in the Asian seabass |
|
| Relations |
| BioSample |
SAMN10442051 |
