GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4572122

Query DataSets for GSM4572122

Status

Public on Jun 23, 2020

Title

5 dpf Untreated liver - clutch 7

Sample type

SRA

Source name

Liver

Organism

Danio rerio

Characteristics

genotype: Tg(fabp10a:ER-tdTomato)
pool: 20
rna qbit concentration(ng/ul): 14.4

Treatment protocol

At 96 hpf, 4 days after the embryos were collected, plates were treated with 1% and 2% Ethanol ( Sigma-Aldrich) for 24 hour exposure with untreated as control. At 5 dpf, 9 am the ethanol was washed out 3X and treated with 500 uM tricaine (Ethyl 3- aminobenzoate methanesulfonate, Sigma-Aldrich, USA). Larvae were sorted for red liver positive and these fish were dissected in 3% methyl-cellulose.

Growth protocol

Mating tanks were set up at approximately 4:30 PM the night before and embryos were collected at 10:30 AM the following morning. Following collection, embryos were divided into 30 mL plates, 60 embryos per plate in egg water at 28C in 10:14 light / dark cycle. Plates were cleaned to remove unfertilized or dead embryos.

Extracted molecule

total RNA

Extraction protocol

Larvae were anesthetized with tricane and 20-35 livers were microdissected for each condition and immediately placed into 500 uL of TRIzol (Thermo Fisher, 15596026).RNA from pooled livers was then extracted per standard TRIzol/Chloroform method and concentrated through isopronanol and resuspended in 20 uL of DNase/RNase free water (Thermo Fisher Scientifc). RNA was quantified by Qubit flurometer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

For library preparation, high quality of total RNA was QCed using a bioanalyzer (Agilent 2100; Agilent Technologies, Santa Clara, CA, USA). Further, mRNA library was prepared using Illumina TruSeq V2 RNA sample Prep Kit (San Diego, CA) according to the manufacturer’s protocol. Briefly, 100 ng of total mRNA was poly-A purified, fragmented, and first-strand cDNA reverse transcribed using random primers. Following second-strand cDNA synthesis, end repair, addition of a single A base, TrueSeq adapter-index was ligated to cDNA libraries, and PCR amplification of 12 cycles was done for enrichment, producing a 350-400 bp fragment including adapters. The fragment sizes and purity of the libraries were confirmed by analyzing on a Bioanalyzer 2100 (Agilent Technologies). The quantities of the libraries required for RNA-seq were determined by real-time qPCR using a KAPA library quantification kit for the Illumina platform (Kapa Bio systems).

Data processing

Illumina Casava1.8 software used for basecalling.
The raw reads were quality assessed using FastQC v0.11.5. The raw reads were then quality trimmed using Trimmomatic,trimmomatic_adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36
Alignments of trimmed reads were performed using tophat2 v2.1.0, with the parameters “–no-novel-junctions” and “–G”
Accepted bam files were used for counting gene read numbers with HTSeq, -a 10 -s no
Test of differential expression of each transposonis was implemented by DESeq2 in Bioconductor
Genome_build: GRCz10
Supplementary_files_format_and_content: .txt Reads count of genes

Submission date

May 27, 2020

Last update date

Nov 04, 2021

Contact name

Kirsten Sadler Edepli

E-mail(s)

[email protected]

Phone

971568327587

Organization name

New York University Abu Dhabi