Illumina Genome Analyzer II sequencing; Functional dissection of QTL-region affecting fat-deposition
Functional dissection of QTL-region affecting fat-deposition
The RNA fragments were then copied into first strand cDNA using reverse transcriptase and random hexamer primers followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. The short cDNA fragments were finally prepared for sequencing on the Illumina Genome Analyzer using reagents provided in the Illumina Genomic DNA Sequencing Sample Prep Kit. The cDNA fragments were end-repaired using T4 DNA polymerase and Klenow DNA polymerase and a single adenine nucleotide base was added to the cDNA fragments by using 3' to 5' exonuclease. This was followed by ligation of Illumina adaptors, gel purification of fragments of approximately 200 bases in size and finally PCR enrichment for 15 cycles. The concentrations of the libraries were measured using Invitrogen Qubit fluorometer and the size and purity were determined using the Agilent DNA 1000 Kit on Agilent 2100 Bioanalyzer. The 89 libraries were diluted in buffer EB from QIAGEN to 10 nM, denaturated with 2 N NaOH to a final DNA concentration of 1.0 nM, diluted to 4 pM with pre-chilled hybridization buffer from Illumina and loaded in individual lanes into a 1.0 mm flowcell together with a single lane of a 2 pM PhiX control library from Illumina.
Experimental Factor: individual
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