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Status |
Public on Nov 03, 2021 |
Title |
TxIB injection after alcohol withdrawal 3 |
Sample type |
SRA |
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Source name |
brain
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Organism |
Danio rerio |
Characteristics |
treatment: TxIB injection after alcohol withdrawal strain: AB developmental stage: adult drug injected: TxIB injection method: retro-orbital injection injected dose: 1 mg/kg, 10 uL per fish tissue: brain
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Treatment protocol |
Lyophilized TxIB was dissolved in normal saline solution (0.9%) and then injected at 10 μL per fish for a final dose of 1 mg/kg using the retro-orbital injection method. Briefly, zebrafish were anesthetized in an ice/water bath within 10 seconds and then removed to a sponge pad for injection. Normal and withdrawal group injected 10 μL normal saline solution per fish to serve as controls. After injection, fish were transferred into freshwater for recovery. Brain tissue dissection were conducted five hours after injection to ensure fish were recovered from anesthesia.
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Growth protocol |
Zebrafish were treated with the escalating alcohol concentration to minimize their mortality. Briefly, zebrafish were first introduced to 0.1% (volume/volume) alcohol-water on the first day, then alcohol concentration was increased by 0.1% every other day, which was 0.2% on the third day. The alcohol concentration reached the top (0.2%) and was held for the next 14 days. From the third week, zebrafish were taken back to the non-alcohol water for 3 hours per day to produce an alcohol withdrawal process. On the 21st day, zebrafish were withdrawn from 0.2% alcohol for the last time before the drug injection and the behavior test. The final concentration of 0.2% was determined through a preliminary experiment to insure the alcohol does not cause a substantial number of deaths during the long-term exposure. Normal group zebrafish were housed in a seperate tank with non-alcohol water.
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Extracted molecule |
total RNA |
Extraction protocol |
Following five hours after drug injection, zebrafish were anesthetized by immersion into the ice-water bath. After cutting the head instantly from the fish body, the brain was dissected from the head and rinsed in the cold PBS solution. The brain tissue was then put into a freezing tube and transferred into the liquid nitrogen immediately. Ten brains were pooled together for one biological replicate and the total RNA was extracted using FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech co., Ltd) following the manufacturer’s instructions. The purity, concentration, and integrity of RNA samples were identified to guarantee the quality of samples for transcriptome sequencing. Library construction was conducted with the following processes: The mRNA was enriched with magnetic beads with Oligo (dT), and randomly interrupted by adding Fragmentation Buffer, then the first cDNA strand was synthesized with six-base random primers (random hexamers) using mRNA as the template, then dNTPs, RNase H and DNA polymerase I were added to synthesize the second cDNA strand and the cDNA was purified using AMPure XP beads. The purified double-stranded cDNA was then terminally repaired, A-tailed, and ligated to sequencing junctions, followed by fragment size selection using AMPure XP beads. Finally, the cDNA library was enriched by PCR.After the library construction, the effective concentration of the library ( > 2nM) was accurately quantified using Q-PCR method to ensure the quality of the library. Then the high-throughput RNA sequencing was performed on an Illumina NovaSeq 6000 sequencer. The sequencing read length was 150 bp paired-end. The clean data of each sample reached 5.79Gb, and the Q30 base percentage was 93.22% and above.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
The adaptor sequences and low-quality sequence reads were removed from the data sets. Raw sequences were transformed into clean reads after data processing. These clean reads were then mapped to the reference genome sequence. Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome. HISAT2 ( 2.0.4) were used to map with reference genome (GRCz11) with the following setting: --dta -p 6 --max-intronlen 5000000. Gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped. Genome_build: GRCz11 Supplementary_files_format_and_content: Tab-delimited text files with raw gene counts and FPKM values for every gene and every sample. N refers to Normal group, S refers to alcohol-withdrawal group, and T refers to TxIB group.
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Submission date |
Nov 01, 2021 |
Last update date |
Nov 03, 2021 |
Contact name |
Kailin Mao |
E-mail(s) |
maokl@hainanu.edu.cn
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Organization name |
Hainan University
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Department |
School of Life Sciences
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Lab |
Key Laboratoryr of Topical Biological Resources of Ministry of Education
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Street address |
Hai Dian Campus of Hainan University, No.58 of Ren Min Road, Mei Lan District, Haikou, Hainan Province,China
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City |
Haikou |
State/province |
Hai Nan |
ZIP/Postal code |
570228 |
Country |
China |
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Platform ID |
GPL24995 |
Series (1) |
GSE186926 |
α6β2* nAChR Antagonist TxIB Changed Gene Expression in Zebrafish (Danio rerio) brain Induced by Alcohol Withdrawal |
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Relations |
BioSample |
SAMN22818868 |
SRA |
SRX12877266 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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