|
| Status |
Public on Sep 24, 2017 |
| Title |
PaternalGrandmother |
| Sample type |
genomic |
| |
|
| Source name |
Blood
|
| Organism |
Bos taurus |
| Characteristics |
cell type: Blood
dna sample: Multi cell
wga: -
|
| Treatment protocol |
The embryos were treated with pronase to dissolve the zona pellucida (0.1% protease from S. griseus, P88110, Sigma-Aldrich in TCM-199 for IVM-IVF and OPU-IVF embryos and 1% for in vivo derived embryos) and were subsequently washed in TCM-199 with 10% FBS followed by Ca+2/Mg+2 free PBS with 0.05% BSA to stimulate blastomere dissociation. Subsequently, the zona-free embryos were washed in Ca+2/ Mg+2 free PBS with 0.1% PVP (wash medium) and were transferred onto a petri dish for blastomere dissociation. Blastomere pick-up and tubing was performed with the use of a mouth-pipetting system using a 75μm capillary.
|
| Growth protocol |
The zygotes of IVM-IVF and OPU-IVF embryos were transferred to synthetic oviductal fluid (SOF) supplemented with essential and non-essential amino acids (SOFaa), 0.4% BSA and ITS (5 µg/ml insulin, 5 µg/ml transferrin and 5 ng/ml selenium). In vitro culture (IVC) occurred in 4-well dishes in 20 µL drops (1 per donor cow) covered with mineral oil. Embryos were incubated at 38.5°C in 5% CO2, 5% O2 and 90% N2. In vivo derived embryos were obtained by flushing of the oviduct
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The isolated blastomeres were placed immediately on dry ice and stored in -20oC for 30 minutes prior to whole genome amplification (WGA). WGA was performed on the day of blastomere isolation. DNA from single blastomeres was whole-genome amplified using commercial MDA kit (REPLI-g Single Cell Kit, Qiagen). MDA amplification of single blastomeres with the single-cell Repli-g kit was performed according to the manufacturer’s instructions for the fast 3-hour protocol. WGA products were purified using SPRI-beads (AMPure) at 0.8x of total reaction volume.
|
| Label |
Cy5/Cy3
|
| Label protocol |
The samples were not labelled before the hybridization. The staining is based on post-hybridization Cy5/Cy3 labelling by extension, according to the manual of Infinium II Assay (Illumina, USA).
|
| |
|
| Hybridization protocol |
According to the manual of Infinium II Assay (Illumina, USA).
|
| Scan protocol |
The scanning of the bead-chips was performed on an iScan, using the iScan Control software (Illumina, USA)
|
| Description |
DNA extracted from a large amount of cells derived from the cow's blood using DNeasy Blood and Tissue kit (Qiagen). Used for subsequent computational workflow and embryo analysis in pedigrees 4757, 4770, 4006, 8301 and 9617.
data structures: The data is provided as five family pedigrees (Cross4757, Cross4006, Cross4770, Cross9617 and Cross8301).
|
| Data processing |
The genotypes, B allele frequencies and LogR values were processed by the Genotyping module of GenomeStudio software. The discrete genotypes were determined using a threshold of 0,75. We subsequently fed these data into siCHILD algorithm, which is equipted with haplarithmisis (Zamani Esteki et al. 2015, AJHG).
|
| |
|
| Submission date |
Feb 24, 2017 |
| Last update date |
Sep 24, 2017 |
| Contact name |
Masoud Zamani Esteki |
| E-mail(s) |
[email protected]
|
| Phone |
+31 43 38 75306
|
| Organization name |
Maastricht University Medical Center
|
| Department |
Clinical Genetics
|
| Lab |
Cellular Genomic Medicine
|
| Street address |
P. Debyelaan 25
|
| City |
Maastricht |
| State/province |
Limburg |
| ZIP/Postal code |
6229 HX |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL21267 |
| Series (1) |
| GSE95358 |
In vitro procedures exacerbate chromosome instability in early cleavage stage embryos |
|
