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Genotyping of CD4+. University of Washington, Stamlab (NREMC)
Genotyping
Project News:
2014/7/14: EXPERIMENT XML file 'NREMC.experiment.genotypingArray.JS-RO-01701.xml' uploaded
alias: JS-RO-01701-genotype
center_name: NREMC STUDY_REF: UwStam_NREMC_CA SAMPLE_DESCRIPTOR: SRS183510 ATTRIBUTES EXPERIMENT_TYPE: Genotyping Array PLATFORM: GPL16104 GROWTH_PROTOCOL: http://roadmapepigenomics.org/protocols TREATMENT_PROTOCOL: None EXTRACT_PROTOCOL: Qiagen DNeasy Blood and Tissue kit (cat #69506). LABEL_PROTOCOL: The chip underwent staining and extension in capillary flow-through chambers. Allele-specific single-base extension of the oligos on the beadchip, using the captured DNA as template, incorporated detectable labels on the beadchip and determined the genotype call for the sample. HYB_PROTOCOL: 200ng of genomic DNA was first denatured and subsequently neutralized to prepare it for amplification. The denatured DNA was then isothermally amplified in an overnight step at 37°C. Next, amplified DNA was enzymatically fragmented for 60 minutes at 37°C using an end-point fragmentation process. The fragmented DNA was then precipitated with isopropanol, allowed to air dry, then resuspended in hybridization buffer. Eight samples were subsequently applied to each HumanOmni2.5-8v1.1 beadchip, which were kept separate with an IntelliHyb seal. The prepared beadchip was incubated overnight in a hybridization oven at 48°C for 16-24 hours with rocking. The amplified and fragmented DNA samples annealed to locus-specific 50mers during hybridization. Following hybridization, unhybridized and non-specifically hybridized DNA was washed away, and the chip was prepared for staining and extension. SCAN_PROTOCOL: Beadchips were scanned using an Illumina iScan+ with ICS v3.2.28 DATA_PROCESSING: Intensity data was extracted with Illumina GenomeStudio software (GenomeStudio v2011.1 with Genotyping Module v1.9.4) with a GenCall cutoff of 0.15. PROBE_GROUP_FILE: Illumina HumanOmni2.5-8v1.1 beadchip META-PROBESET_FILE: None VALUE_DEFINITION: None |
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