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| Status |
Public on Sep 24, 2017 |
| Title |
E02_Bl005_Cross4770 |
| Sample type |
genomic |
| |
|
| Source name |
Blastomere
|
| Organism |
Bos taurus |
| Characteristics |
cell type: Blastomere
dna sample: Single Cell
wga: MDA (Repli-G Single Cell kit)
|
| 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).
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| |
|
| 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 single blastomere derived from cleavage stage OPU-IVF embryo of pedigree 4770 and subsequently whole genome amplified by Repli-G Single cell kit according to manufacturer's 3h protocol.
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).
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| |
|
| Submission date |
Feb 24, 2017 |
| Last update date |
Sep 24, 2017 |
| Contact name |
Masoud Zamani Esteki |
| E-mail(s) |
masoud.zamaniesteki@mumc.nl
|
| 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 |
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