|
| Status |
Public on Apr 13, 2018 |
| Title |
Zebrafish 2 hpf whole embryo injected with ConMO (Exp. ID: rs1a) |
| Sample type |
SRA |
| |
|
| Source name |
Zebrafish whole embryo
|
| Organism |
Danio rerio |
| Characteristics |
hour post fertilization: 2
morpholino: Control
strain: AB
|
| Treatment protocol |
Approximately 1 nl of the solution containing TUT4/7 or control morpholinos was injected into wild-type zebrafish embryos at 1-cell stage.
|
| Growth protocol |
Zebrafish embryos were obtained by the natural mating of wild-type AB strain and grown in embryo medium at 28.5˚C. Embryos were staged according to standard morphological criteria.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each sample, embryos were collected and total RNA was extracted with TRIzol (Invitrogen). The samples were spiked in with ERCC RNA Spike-In Mix (Ambion) for normalization purpose. The quality of total RNA was checked by Agilent Bioanalyzer 2100 (Agilent).
Ribosomal RNA was depleted from total RNA using Ribo-Zero Gold kit (Epicentre). RNA- seq libraries were constructed using TruSeq stranded total RNA library prep kit (Illumina) and sequenced in Illumina Nextseq 500 v2 to yield paired-end 76nt reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
rs1a-ConMO-2hpf
|
| Data processing |
The sequences were basecalled by Illumina RTA embedded in NextSeq Control Software 1.3.2 by the standard workflow.
Sequenced reads from RNA-seq data were mapped to the GRCz10 genome with RefSeq release 104 annotations above using STAR 2.5.3a via RSEM 2.1.31.
The gene-level quantifications were performed using RSEM with the default options for strand-specific and paired-end RNA-seq. The "rs1a" set of our libraries was between-sample normalized using RUV-seq 1.10.0. The standard workflow suggested by RUV-seq did not work due to the global changes of maternal transcripts following the progression of embryogenesis. Instead, we first chose a subset of spike-in RNAs that have ≥ 256 “expected counts” of reads in at least 90% of samples. Then, the counts were normalized by the RUVg algorithm (k = 1). The geometric means of the normalized counts were used as size factors for the scale normalization of all other RNAs. The ERCC spike-in reads in our second RNA-seq dataset were too unreliable to be used as references. Even the raw read counts mappable to the reference spike-in sequences were significantly out of the linear correlations between any two samples. As a workaround, we picked the internal controls from the first set to find genes that express more than 500 normalized reads with ≤ 0.2 of the log2 largest difference between two samples. Fourteen genes satisfied the criteria to become a set of internal controls. The internal reference genes were used in place of spike-in RNAs for processing counts by the modified indirect RUVg approach.
Genome_build: GRCz10
Supplementary_files_format_and_content: comma-separated text file include RUVg-normalized (rs1a and rs1b) or TPM-normalized (rs2) values for each sample.
|
| |
|
| Submission date |
Feb 26, 2018 |
| Last update date |
Apr 13, 2018 |
| Contact name |
Hyeshik Chang |
| E-mail(s) |
[email protected]
|
| Organization name |
Seoul National University
|
| Department |
School of Biological Sciences
|
| Lab |
Hyeshik Chang Lab
|
| Street address |
Building 203 Room 525, School of Biological Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu
|
| City |
Seoul |
| State/province |
South Korea |
| ZIP/Postal code |
08826 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL20828 |
| Series (1) |
| GSE111152 |
Maternal mRNA degradation by TUT4 and TUT7 in the zebrafish maternal-to-zygotic transition |
|
| Relations |
| BioSample |
SAMN08611111 |
| SRA |
SRX3744210 |
