|
| Status |
Public on Dec 15, 2017 |
| Title |
FGL_T |
| Sample type |
SRA |
| |
|
| Source name |
left gonad
|
| Organism |
Gallus gallus |
| Characteristics |
tissue: gonad
position: left
Sex: female
substance: Tributyltin 10 pg SN/g BW
developmental stage: E 19
|
| Treatment protocol |
Gonads were immediately stored at -80 degrees C in RNA lysis buffer until RNA isolation.
|
| Growth protocol |
Chicken embryos were bred 19 days until section in a ThermoStar 100 egg incubator. Incubation was performed at 37.6 degrees Centigrade and 60% humidity, eggs were turned once every 2 hours. Embryos were decapitated 2 days before anticipated hatching and dissected under a microscope. Gonads were extracted and separated from embedding tissue.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from right and left gonads (ten individuals of each group) was extracted with the SV Total RNA Isolation System Kit according to manual 048 (Promega). Deviating from the protocol, on-column DNAseI digestion of genomic DNA was elongated from original 15 min to 30 min. A second DNaseI digestion of genomic DNA was carried out in solution. Sequencing was performed on Illumina's Genome Analyzer Iix, subsequent base calling carried out by Illumina's GAPipeline.
DNase-treated total RNA was subjected to oligo(dT)-based reverse transcription using SuperScript III Reverse Transcriptase (Invitrogen) and an anchored, 5’ biotinylated oligo(dT) primer comprising the recognition site for EcoP15I. 3’ cDNA fragments were enriched via binding to streptavidin-coated paramagnetic beads (Dynabeads M-270 Streptavidin, Invitrogen) subsequent to cleavage of 5’-CATG sites in reverse-transcribed cDNAs by NlaIII (NEB). An adaptor comprising a known barcoding sequence and a second recognition site of EcoP15I was ligated to the enriched fragments using T4 DNA Ligase (Fermentas). The adaptor-ligated cDNA fragments were released from the beads via digestion by EcoP15I (NEB), end-repaired by KOD DNA Polymerase (Blunting High Kit, Toyobo), and subsequently ligated to a second barcoding adaptor using the T4 Ligase reagent provided with the Ligation high Ver. 2 Kit (Toyobo). Barcoding sequences of the adaptor-ligated cDNA fragments were extended during subsequent PCR-amplification employing Phusion Hot Start High-Fidelity DNA Polymerase (Fermentas) according to the manufacturer’s recommendations.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Library strategy: SuperSAGE
GAPipeline1.4.0
Raw data was analyzed with GenXPro's SuperSAGE data processing pipeline. Distinct libraries were sorted out from the bulked sequencing data according to their respective indices.
Followed by elimination of PCR-derived tags identified by TrueQuent technology. 26bp SuperTAGSs were extracted from the remaining sequences.
The remaining reads were mapped on the Gallus gallus genome Galgal4 from ENSEMBL with novoalign allowing one mismatch.
Genome_build: Gallus_gallus.Gallus_gallus-4.0
Supplementary_files_format_and_content: Microsoft Excel Table with TPM values and statistics
|
| |
|
| Submission date |
Dec 15, 2017 |
| Last update date |
Dec 18, 2017 |
| Contact name |
GenXPro GenXPro |
| Organization name |
GenXPro GmbH
|
| Street address |
Altenhöferallee 3
|
| City |
Frankfurt am Main |
| ZIP/Postal code |
60438 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13797 |
| Series (1) |
| GSE108141 |
Morphological and transcriptomic effects of endocrine modulators on the gonadal differentiation of chicken embryos: the case of tributyltin (TBT) |
|
| Relations |
| BioSample |
SAMN08182006 |
| SRA |
SRX3474129 |
