|
| Status |
Public on May 15, 2015 |
| Title |
S1_ebv05_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 |
Cultured cells were washed with phosphate buffered saline (PBS) solution to completely remove medium traces prior to the single cell isolation.
|
| Growth protocol |
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.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Single-cell DNA was amplified following: (i) an MDA approach with Genomi Phi V2 (GE Healthcare, Piscataway, NJ). 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. (ii) A PCR-based approach with PicoPlex (Rubicon Genomics, MI, USA). 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.
|
| Label |
biotin
|
| Label protocol |
according to Affymetrix GeneChip Mapping 500K Assay Manual
|
| |
|
| Hybridization protocol |
according to Affymetrix GeneChip Mapping 500K Assay Manual
|
| Scan protocol |
according to Affymetrix GeneChip Mapping 500K Assay Manual
|
| Description |
Single cell from an Epstein-Barr virus transformed lymphoblastoid cell line derived from sibling S1.
|
| Data processing |
All the DNA samples were genotyped using the dynamic model (DM) algorithm embedded in the ‘GeneChip Genotyping Analysis Software (GTYPE) version 4.1 (Affymetrix)’ using a homozygous and heterozygous calling threshold of 0.12, (2) the BRLMM algorithm within the ‘Genotyping console 3.0.1’ software (Affymetrix) using a scoring threshold of 0.1 and (3) the Birdseed algorithm using both 'birdseed-v1' and 'birdseed-dev' versions. The latter is the most recently incorporated development version of birdseed from The Broad Institute (http://www.broadinstitute.org/mpg/birdsuite/birdseed.html).
The CEL files are provided online, and DM-determined genotypes are provided in the 'NspMatrix' sheet.
|
| |
|
| Submission date |
Aug 28, 2014 |
| Last update date |
May 16, 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 |
GPL3718 |
| Series (2) |
| GSE60907 |
Concurrent whole-genome haplotyping and copy number profiling of single cells (Affymetrix) |
| GSE60910 |
Concurrent whole-genome haplotyping and copy number profiling of single cells |
|
