|
| Status |
Public on Dec 21, 2019 |
| Title |
28 hpf miR-34a-/- treated with 1μM CPT rep2 |
| Sample type |
SRA |
| |
|
| Source name |
whole embryo
|
| Organism |
Danio rerio |
| Characteristics |
genotype: miR-34a-/-
Stage: 28 hpf
treatment: 4 hours with 1μM camptothecin
|
| Treatment protocol |
Camptothecin (CPT) treatments were performed by adding either DMSO (control groups) or the 2 mM stock CPT solution in DMSO to fish water for 1 µM concentration and incubating embryos for 4 hours.
|
| Growth protocol |
Zebrafish experiments and husbandry follows standard protocols in accordance with the standards of the University of Ottawa Animal Care Committee (approval number: CHEOe-3168-A1). Zebrafish embryos were maintained at 28.5 °C during development and as adults. Embryos were grown in egg water (1x E3 prepared from 60x E3 stock).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Each total RNA sample was extracted from 30-50 zebrafish embryos by homogenizing them in 500 µL Trizol reagent (Thermo Fisher Scientific, 15596026) using 1 mL syringe and 22G needle. Lysates were centrifuged at 12,000 g at 4ºC for 5 minutes and the supernatant was transferred to Phasemaker Tubes (Thermo Fisher Scientific, A33248) and RNA was purified according to the Phasemaker Tubes protocol manual.
Total RNA samples were measured and analyzed for integrity on Agilent Tape Station. Samples with RNA integration number ≥8 were selected for library preparation. Poly-A enrichment was performed from 20 µg of total RNA to enrich for mRNA (Dyna beads mRNA direct micro Kit). 100ng of poly-A enriched RNA was fragmented using RNase III and purified using magnetic bead clean up module (RNA Seq V2 kit, Life Technologies). The size distribution of the fragmented RNA was assessed on Agilent Tape Station using RNA HS screen tape assay and 50 ng of fragmented polyA-enriched RNA was used to prepare whole transcriptome library (RNA seq V2). Yield and size distribution of the library was analyzed on Agilent Tape Station using D1000 screen tape. Barcoded library was equally pooled and amplified onto Ion Sphere™ Particles (ISPs) from Ion Pi HiQ OT2 kit (Life Technologies). ISPs enriched with template library were loaded onto Ion PI chip V3 and sequenced on Ion Proton from Thermo Fisher.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Data processing |
Read trimming: the raw reads had the adapters “ATCACCGAC” removed and filtered by the quality of 20 with the Trim Galore (v0.4.4).
Read mapping: The reads passed through the two-step mapping for RNA-Seq data from Ion proton: first with STAR (v2.7) and then mapping the unmapped reads from the first alignment using bowtie2. The two mapped files were merged with Picard tools (v2.5.0).
Read counting: The counts were extracted using HTSeq (V0.9.1).
Genome_build: danRer11
Supplementary_files_format_and_content: read counts
|
| |
|
| Submission date |
Dec 20, 2019 |
| Last update date |
Dec 22, 2019 |
| Contact name |
Sergey 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 |
ON |
| ZIP/Postal code |
K1N 9B4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL24059 |
| Series (1) |
| GSE142440 |
RNA-seq analysis of gene regulation by miR-34a and expression changes induced by DNA damage in zebrafish |
|
| Relations |
| BioSample |
SAMN13656608 |
| SRA |
SRX7423117 |
