 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 11, 2020 |
| Title |
mondoa_s_3 |
| Sample type |
SRA |
| |
|
| Source name |
total RNA
|
| Organism |
Danio rerio |
| Characteristics |
tissue: embryo
genotype: MZmondoA mutant (allel: ka405, ZFIN ID: ZDB-ALT-180628-2)
phenotype: affected epiboly
Stage: 5 hpf
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Embryos were sampled in liquid nitrogen and stored at -80°C until further processing. RNA was extracted with TRIzol (Life Technologies) following the manufacturer’s instructions with minor modifications: 30 larvae were sampled in a volume of 1.5 ml TRIzol reagent. Samples were kept at least overnight at -80°C. In order to disrupt the cell membranes, the samples were passed four times through a syringe with a diameter of 0.45 mm.
Total RNA-Seq libraries were generated from 500 ng of total RNA using TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, cytoplasmic and mitochondrial ribosomal RNA (rRNA) was removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the depleted RNA was fragmented into small pieces using divalent cations at 94oC for 2 minutes. Cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
counts_exon.txt, counts_intron.txt
|
| Data processing |
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Sequences were aligned to the full reference genome index build with Ensembl annotation to improve accuracy on splices junctions using STAR 2.4.0i
A custom Perl script was used to quantify uniquely mapped reads. For each Ensembl gene, all exons of the respective annotated protein-coding transcripts were considered.
Genome_build: zv10
Supplementary_files_format_and_content: counts_exon.txt, exonic counts for each indicated gene and sample
|
| |
|
| Submission date |
May 28, 2020 |
| Last update date |
Jul 12, 2022 |
| Contact name |
Benjamin D Weger |
| Organization name |
EPFL
|
| Department |
SV
|
| Lab |
Naef Lab
|
| Street address |
Station 15
|
| City |
Lausanne |
| State/province |
VD |
| ZIP/Postal code |
76344 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21741 |
| Series (2) |
| GSE134778 |
RNA-seq of mondoa zebrafish mutants |
| GSE144350 |
Regulation of cholesterol biogenesis by the glucose-sensing transcription factor MondoA is required for zebrafish epiboly |
|
| Relations |
| BioSample |
SAMN15049703 |
| SRA |
SRX8419562 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
