|
| Status |
Public on Mar 10, 2021 |
| Title |
78 hpf liver from transgenics overexpressing nAtf6 - Clutch 1 |
| Sample type |
SRA |
| |
|
| Source name |
Liver
|
| Organism |
Danio rerio |
| Characteristics |
genotype: Tg(fabp10a:CAAX-EGFP); Tg(fabp10:nAtf6-mcherry; cmlc2:EGFP)-C
pool: 20
rna qbit concentration(ng/ul): 11
|
| Treatment protocol |
At approximately 78 hpf/3 dpf - 3 PM and at 120 hpf/ 5 dpf- 9 AM, plates were treated with 500 uM tricaine (Ethyl 3- aminobenzoate methanesulfonate, Sigma-Aldrich, USA) and were first sorted for green liver positive as all fish needed to have marked livers to proceed. Next, green liver positive fish were spilt between siblings without green hearts (larvae that don't express nAtf6 - Sample name- Sibs) and with green hearts (larvae that express nAtf6- Sample name : Natf6). These fish were dissected in 3% methyl-cellulose.
|
| Growth protocol |
This experiment was conducted by natural spawnings between tg:(fabp10:nAtf6-cherry; cmlc2:GFP)-C and tg(fabp10a:CAAX-EGFP ) . 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 embryo medium (Zebrafish Information Network). Plates were cleaned daily to remove unfertilized or dead embryos.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
20 livers were microdissected 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.(Thermo Scientific) The 120 hpf samples were DNAseI treated for 30 minutes at 37°C followed by RNA purification (RapidOut DNA Removal Kit–Thermo Fisher Scientific).
|
| |
|
| 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). The samples collected at 120 hpf were rRNA depleted by Ribo Zero Gold treatment. Further, mRNA library of all samples 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 Casava software used for basecalling.
Sequencing quality was assessed by using MultiQC v1.7
Alignment by HISTA2 with default parameters, only paired reads are aligned and multiple alignments are kept
Gene expression is counted in the exon of ensemble gene annotation with HTseq, in union mode
Test of differential expression of Genes or transposonis was implemented by DESeq2 in Bioconductor V4.0.2
Genome_build: GRCz10
Supplementary_files_format_and_content: csv files for raw reads count of genes
|
| |
|
| Submission date |
Mar 09, 2021 |
| Last update date |
Nov 04, 2021 |
| Contact name |
Kirsten Sadler Edepli |
| E-mail(s) |
kirsten.edepli@nyu.edu
|
| Phone |
971568327587
|
| Organization name |
New York University Abu Dhabi
|
| Department |
Biology
|
| Street address |
PO Box 129188
|
| City |
Abu Dhabi |
| State/province |
Abu Dhabi |
| ZIP/Postal code |
000 |
| Country |
United Arab Emirates |
| |
|
| Platform ID |
GPL25922 |
| Series (2) |
| GSE130800 |
Transcriptomic profiling of zebrafish livers with nAtf6 overexpression at collected at 78 and 120 hpf |
| GSE130801 |
Transcriptomic profiling of zebrafish livers at 5 dpf with nAtf6 overexpression or in response to ethanol exposures |
|
| Relations |
| BioSample |
SAMN18221856 |
| SRA |
SRX10292189 |
