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Sample GSM4644658

Query DataSets for GSM4644658

Status

Public on Dec 16, 2020

Title

A2-12_S18

Sample type

SRA

Source name

plasma sample

Organism

Solea senegalensis

Characteristics

Sex: Male
tissue: Plasma
treatment: Untreated
sampling point: 2d

Treatment protocol

The fish maintained in two of the four tanks (n = 31 fish) were muscularly injected with KISS2 decapeptide at a dose of 250 µg/kg body weight, based on previous reports of positive results of KISS2-10 eliciting gonadotropin release and gonadal development. The remaining fish (n = 31) were injected with phosphate buffered saline (PBS) to test the placebo effect and thus to be used as a control group. The same day, blood samples were collected 2 (2h) and 4 (4h) hours after treatment, as previously described, to determine the acute effect on hormonal levels. Further samplings were performed 2 (2d) and 4 (4d) days after the treatments, to collect blood. At this stage, also plasma was collected for detection of microRNAs. For that, samples of blood (up to 2 mL) were collected from each fish and deposited in a 2 mL Eppendorf tube previously treated with cold buffered sodium citrate (3.8 % in 0.01 M PBS), as well as the syringe. After 15 min at 4 °C, plasma was separated from other blood components through differential centrifugation (15 min at 3000 g) and collected. No visible sign of haemolysis was noted in collected plasma samples. A total of 100 μL of plasma was sampled to perform sex hormone quantification and stored at -20 °C until steroid extraction. The remaining plasma was re-centrifuged at 3000 g for 5 min (to avoid cell debris contamination) and 500 μL of supernatant plasma was collected, snap-frozen in liquid nitrogen, and stored at -80 °C until RNA isolation and analysis.

Extracted molecule

total RNA

Extraction protocol

SncRNAs were isolated from blood plasma samples using miRNeasy Serum/Plasma Kit (Qiagen, Germany) following manufacturer instructions, and assessment of RNA quality and quantity was performed in a 2200 TapeStation Nucleic Acid system using High Sensitivity RNA ScreenTapes (Agilent, USA). Library preparation of 30 samples were performed using NEXTflex Small RNA-Seq kit v3 (Bioo Scientific, USA) for Illumina platforms following manufacturer protocol, and libraries size, purity, and concentration were evaluated on a High Sensitivity D1000 ScreenTape (Agilent, USA). Normalized libraries were clustered in one pool and multiplexed sequencing was done in two independent runs (15 libraries per run) by the NextSeq500 sequencer using a NextSeq High Output kit v2 (75 cycles) (Illumina, San Diego, CA), following the protocol provided by the manufacturer.
NEXTflex Small RNA-Seq kit v3 (Bioo Scientific, USA)

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina NextSeq 500

Data processing

Adapter removal: Cutadapt 2.1
Mapping: Bowtie2 v2.4.1
Annotation: Htseq-count 0.6.0

Submission date

Jun 29, 2020