|
| Status |
Public on May 15, 2015 |
| Title |
S1_ebv04_picoplex |
| Sample type |
genomic |
| |
|
| Source name |
EBV-transformed lymphoblast
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: EBV-transformed lymphoblast
dna sample: Single cell
wga: PicoPlex
hybridization protocol: Conventional
|
| Treatment protocol |
The embryos that were not suitable for transfer on day 4 were dissociated and single blastomeres were collected for further analysis. The zona pellucida was removed by incubation in a droplet of Tyrode's solution (Sigma-Aldrich, USA). The acid was and consequently washed away and the embryos were dissociated in a droplet of Ca2+/Mg2+ free medium (Life Global, Canada).
|
| Growth protocol |
The embryos were in vitro cultured in Life Global medium (Life Global, Canada) or Quinns Advantage Protein Plus Medium (Cooper Surgical, USA). On day 2 and 3 after fertilization, embryo development was evaluated for the number of blastomeres, the percentage of fragmentation and the symmetry of the blastomeres. All ≥ 6 cell stage embryos that had less than 25% fragmentation on day 3 after fertilization were biopsied using a non-contact, 1.48 µm diode laser system (Fertilase®; MTG, Germany) coupled to an inverted microscope, after first being incubated in Ca2+/Mg2+ free medium. One or two blastomeres were gently aspirated from each embryo for the conventional FISH- or PCR-based PGD. The embryos were immediately transferred to fresh medium; while the aspirated blastomeres were separately washed twice with Ca2+/Mg2+ free medium to remove possible oil remnants.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Single cells were washed in 1xPBS and transferred into a 200 µl PCR tube containing 1.5 µl alkaline lysis buffer (ALB; 200 mM KOH and 50 mM DTT) by mouth controlled pipetting using 75 µm stripper tips (Origio, Denmark). The samples were stored at -20°C for at least 30 min and were further incubated for 10 min at 65°C prior to the multiple displacement amplification (MDA) reaction. Single cell DNA was amplified following an MDA-approach with GenomiPhiV2 (GE Healthcare, USA). The MDA products were purified using the High Pure PCR Product Purification Kit (Roche, Switzerland) and resuspended in 70 µl of elution buffer. All amplification products were quantified with a spectrophotometer (Nanodrop ND-1000 spectrophotometer). Single-cell amplifications yielding less than 2 µg DNA were not further used.
|
| 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), with the exception that the fragmentation step was shortened from 20-24h to 7h and the hybridization step was shortened from 16-20h to 6h for the rapid protocol.
|
| Scan protocol |
The scanning of the bead-chips was performed on an iScan, using the iScan Control software (Illumina, USA)
|
| Description |
Single cell from an Epstein-Barr virus transformed lymphoblastoid cell line derived from sibling S1. EBV-transformed lymphoblastoid cells were cultured in 75-cm2 plastic flasks (BD Falcon, USA) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (Gibco, USA) medium supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Scientific, USA) at 37oC, 5% CO2. Single cells were washed in 1xPBS and transferred into a 200 µl PCR tube containing 1.5 µl PBS by mouth controlled pipetting using 75 µm stripper tips (Origio, Denmark). The samples were further whole genome amplified using PicoPLEX (Rubicon Genomics, USA), according to the protocol of the manufacturer. The WGA products were purified using the High Pure PCR Product Purification Kit (Roche, Switzerland) and resuspended in 70 µl of elution buffer. All amplification products were quantified with a spectrophotometer (Nanodrop ND-1000 spectrophotometer; Nanodrop Technologies). Single-cell amplifications yielding less than 2 µg DNA were not further used. The sample was processed according to the manual of Infinium II assay (Illumina, USA).
|
| Data processing |
The genotypes, B allele frequencies and LogR values were extracted by the Genotyping module of GenomeStudio software. The discrete genotypes were determined using a threshold of 0,75 in GenCall.
|
| |
|
| Submission date |
Aug 28, 2014 |
| Last update date |
May 15, 2015 |
| 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 |
GPL13829 |
| Series (2) |
| GSE60909 |
Concurrent whole-genome haplotyping and copy number profiling of single cells (Illumina) |
| GSE60910 |
Concurrent whole-genome haplotyping and copy number profiling of single cells |
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