|
| Status |
Public on Jan 16, 2014 |
| Title |
(-)4SUTP_256cell |
| Sample type |
SRA |
| |
|
| Source name |
embryo, 256-cell stage, unlabeled
|
| Organism |
Danio rerio |
| Characteristics |
strain: AB
genotype: wild-type
tissue: embryo
developmental stage: 256-cell
4-thio utp labeling: unlabeled
replicate: replicate 1
|
| Treatment protocol |
Embryos were microinjected at the 1-cell stage with 1 nl of 50 mM 4-thio-UTP in 10 mM Tris-HCl pH 7.4 (Ambion).
|
| Growth protocol |
Zebrafish WT AB strain was maintained and raised under standard conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from staged embryos was extracted with Trizol, biotinylated with EZ-Link HPDP-Biotin and subsequently purified with streptavidin-coated magnetic beads to isolate newly transcribed RNA. Labeled and unlabeled samples were treated in the same way.
Libraries were prepared for the HiSeq 2000 machine (Illumina). Briefly, RNA was amplified with the WT-Ovation™ One-Direct RNA Amplification System (NuGEN). The reaction was stopped after the SPIA amplification and double-stranded cDNA with random Hexamers created. Double-stranded cDNA was sheared to ~200-300bp fragment size, end-repaired with the NEBNext End Repair Module (NEB) and the resulting fragments were adenylated at the 3'-end with the NEBNext dA-Tailing Module (NEB).TruSeq adapters were ligated and the libraries were PCR amplified for 15 cycles. The concentration of molecules with adapters ligated in the final library was determined by qPCR with the KAPA Library Quant Kit (Kapa Biosystems).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
processed data file: GSE47709_comb_genes_2.tsv
|
| Data processing |
Basecalling with Illumina Casava 1.7 software.
Overrepresented reads (adapter, SPIA-amplification primers) were identified from FastQ output and trimmed as described in Kircher M., Methods Mol Biol, 2012.
Reads were mapped to the zebrafish genome assembly Zv9/GCA_000002035.2 with TopHat 1.3.3 using the following parameters --butterfly-search --library-type=fr-unstranded --splice-mismatches=1 --max-multihits=100
Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated using Cufflinks 2.0.2 using the Ensembl release 64 with the following parameters: --multi-read-correct --max-bundle-frags=20000000 --library-type=fr-unstranded
Only transcripts or genes with a FPKM value larger than the width of the 95% confidence interval (FPKM > Δ95 FPKM) were considered to be expressed as described in Nagaraj et al., Mol Syst Biol., 2011.
Technical replicates of (+)4SUTP samples were pooled, mapped and sampled to the same number of reads per developmental time points, genes/transcripts had to be expressed in two of the 3 time points.
The sum of FPKM values for protein-coding mitochondrial transcripts/genes was assumed to be constant and used to normalize FPKM values for pooled (+)4SUTP samples between developmental time points.
For (-)4SUTP to (+)4SUTP comparison, reads from all samples were sampled to 8775274 before FPKM calculation.
Genome_build: Zv9
Supplementary_files_format_and_content: Tab-delimited text files include Ensembl genes/transcripts and FPKM values for each sample.
|
| |
|
| Submission date |
Jun 06, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Patricia Heyn |
| Organization name |
Max Planck Institute of Molecular Cell Biology and Genetics
|
| Street address |
Pfotenhauerstrasse 108
|
| City |
Dresden |
| ZIP/Postal code |
01307 |
| Country |
Germany |
| |
|
| Platform ID |
GPL14875 |
| Series (1) |
| GSE47709 |
Identification of zygotically transcribed genes in zebrafish by 4-thio-UTP metabolic labeling |
|
| Relations |
| BioSample |
SAMN02191951 |
| SRA |
SRX297178 |
