Cbot and hiseq paired end cluster kit version 3

DNAs from each sorted tumor population and a patient matched control sample were sequenced within the Mayo Clinic Medical Genome Facility (MGF) using established protocols for whole exome analysis. Briefly, whole exon capture was carried out with Agilent’s SureSelect Human All Exon 71 MB v6 kit. 500 ng of the prepped library is incubated with whole exon biotinylated RNA capture baits supplied in the kit for 24 hours at 65°C. The captured DNA:RNA hybrids are recovered using Dynabeads MyOne Streptavidin T1 (Dynal). The DNA was eluted from the beads and desalted using purified using Ampure XP beads (Agencourt).The purified capture products were then amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. Libraries were loaded onto paired end flow cells at concentrations of 4–5 pM to generate cluster densities of 600,000–800,000/mm² using the Illumina cBot and HiSeq Paired end cluster kit version 3.The flow cells are sequenced as 101 X 2 paired end reads on an Illumina HiSeq 2500 or 4000 using TruSeq SBS sequencing kit version 3 and HiSeq data collection version 1.4.8 software. Base-calling was performed using Illumina’s RTA version 1.12.4.2.