|
| Status |
Public on Sep 29, 2017 |
| Title |
48 hpf control MO-injected sample A |
| Sample type |
SRA |
| |
|
| Source name |
whole embryos
|
| Organism |
Danio rerio |
| Characteristics |
morpholino: control
strain: Tubingen
Stage: 48 hpf
|
| Treatment protocol |
The hace1-HECT (5’-CCCTCGAACTGTTAGACAGAATAAA-3’) and standard control morpholinos (5’-CCTCTTACCTCAGTTACAATTTATA-3’) were purchased from Genetools LLC (Philomath, OR). The hace1 morpholino splice site resides within the catalytically active HECT domain, upstream of the critical cysteine residue required for hace1 ubiquitin ligase function. Knockdown of hace1 was verified by RT-PCR as described in Daugaard et al (Daugaard et al., 2013).
|
| Growth protocol |
Zebrafish housing, breeding conditions, and developmental staging of larvae, were performed according to Westerfield (Westerfield, 2000). Use of zebrafish in this study was approved by the Dalhousie University Committee on Laboratory Animals (Protocol 15-123). All zebrafish embryos were maintained in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) in 10 cm Petri dishes at 28oC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol Reagent RNA Extraction protocol (Ambion RNA Life technologies) was used to extract RNA from pooled samples of hace1 or control morphants (n=6 per group). Approximately 100 embryos per sample were used. Tubingen wild type strain (TU) fish were previously injected with either hace1 MO or control MO using the injection protocol noted above. RNA samples were quantified using the NanoDrop Spectrophotometer ND-1000 (Thermo Fisher Scientific, Wilmington, DE). RNA yields for this experiment were lower than the 100µg of RNA needed for RNA sequencing, so 3 samples were pooled into one, for a final n=2 for each of the hace1 MO and control MO groups.
Barcoded cDNA libraries were prepared using RNA-Seq Version 2 kit from Life Technologies following their recommended protocol. Library preparations were assayed for both quality control and quantity using Experion DNA 1K chip (Life Technologies) and diluted to 16 pM concentration. Samples were sequenced using a Proton Sequencer from Life Technologies on a PI chip following the manufacturer recommended protocol. Each chip was loaded with 2 samples.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Data processing |
Torrent_Suite 4.2.1 used for basecalling
Trimming with Cutadapt v1.2.1
Second pass trimming with home-made script in order to remove reads shorter than 17bp and larger than 300bp and Q<20
2 steps alignement for RNA with tophat v2.0.6 and bowtie2 v2.0.2
Quantification and normalization with StrandNGS Version 2.1 with the following settings
Strand specific reads: false
Consider partial reads: true
Max EM Iterations: 1000
Convergence criterion for EM: 0.0010
Normalization Type = DeSeq
Threshold for normalization = 1
Baseline type = median of all samples
Genome_build: Danio_rerio_Zv9-78 Transcripts (2015.01.22)
Supplementary_files_format_and_content: tab-delimited text files include normalised values or raw counts for each sample
|
| |
|
| Submission date |
Sep 28, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Sergey V Prykhozhij |
| E-mail(s) |
[email protected]
|
| Phone |
9028178846
|
| Organization name |
CHEO Research Institute/University of Ottawa
|
| Lab |
Berman Lab
|
| Street address |
Room 318/319 | CAREG, 20 Marie-Curie Private
|
| City |
Ottawa |
| State/province |
Ontario |
| ZIP/Postal code |
K1N 9B4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL24059 |
| Series (1) |
| GSE104380 |
Analysis of gene expression changes by knock-down of hace1 tumour suppressor in zebrafish |
|
| Relations |
| BioSample |
SAMN07715685 |
| SRA |
SRX3228185 |
