The isolated samples are stored in -80deg C deep freezer.
Growth protocol
Peripheral blood drawn from venous was stored in an icebox after obtaining to keep cool and was centrifused within one hour. After discarding supernatant, the blood was isolated as serum, plasma, RBC, whole blood, and buffy coat and stored in cryotube.
Extracted molecule
genomic DNA
Extraction protocol
After the blood samples are thawed, standard citrate buffer is added, mixed, and the tubes are centrifuged and the top portion of the supernatant is discarded (twice). The pellet is resuspended in a solution of SDS detergent and proteinase K, and the mixture is incubated at 55deg C for one hour. The sample then is phenol extracted once with a phenol/chloroform/isoamyl alcohol solution, and after centrifugation the aqueous layer is removed to a fresh microcentrifuge tube. The DNA is ethanol precipitated, resuspended in buffer, and then ethanol precipitated a second time. Once the pellet is dried, buffer is added and the DNA is resuspended by incubation.
Label
Biotin
Label protocol
About 200ng of genomic DNA is amplified and randomly fragmented into 25 to 125 base pair (bp) fragments. gDNA initial amplification reacted in 40㎕ reaction volume, containing 20㎕ volume of genomic DNA at a concentration of 10ng/㎕, 20㎕ of Denaturation Master Mix. The reaction of initial amplification carried out as follows: 10min at Room Temperature. After the initial amplification, the incubated products were amplicated with 130㎕ of Axiom 2.0 Neutral Soln, 225㎕ Axiom 2.0 Amp Soln and 5㎕ Axiom 2.0 Amp Enzyme. The amplification reactions were carried out as follows: 23hour ± 1hour at 37℃. The amplification products were performed in optimized reaction to amplify fragments between 200-1,100 base pairs. A fragmentation step then reduced the amplified products to segments of approximately 25-50 bp, which were then end-labeled using biotinylated nucleotides.
Hybridization protocol
Following hybridization, the bound target is washed under stringent conditions to remove non-specific background to minimize background noise caused by random ligation events. Each polymorphic nucleotide is queried via a multi-color ligation event carried out on the array surface.
Scan protocol
After ligation, the arrays are stained and imaged on the GeneTitan MC Instrument (Affymetrix, Santa Clara, CA, USA)
Description
Hybridized to Axiom®_Exome_319
Data processing
Using rawdata(cel files) as input, data analysis was conducted by Affymetrix power tools(APT) in linux server. APT-probeset-gneotype program, one of the APT applications, was used to gain genotype call as well as CHP files, which was run by birdseed-v2 algorithm as analysis option.