Quick dna 96 plus kit

Whole exome sequencing (WES) was performed as previously described (21 (link)). DNA from flash-frozen tumor samples was extracted using the Quick-DNA 96 Plus kit (Zymo). The Agilent SureSelect Human All Exon 50-Mb target enrichment kit v4 was used to capture all human exons for deep sequencing using the vendor’s protocol v2.0.1. The SureSelect Human All Exon Kit targets regions of 50 Mb in total size, which is approximately 1.7% of the human genome. In brief, 3 μg genomic DNA was sheared with a Covaris S2 to a mean size of 150 bp. Five hundred nanograms of library DNA was hybridized for 24 h at 65 °C with the SureSelect baits. 15 ng final exome-enriched library (without barcode) was used as a template in a 50-μl PCR reaction. The Herculase II Fusion enzyme (Agilent) was used together with the NEBNext Universal PCR primer for Illumina and NEBNext Index primer (NEB #E7335S) under the following conditions: the initial denaturation step for 2 min at 98 °C was followed by four cycles of 30s at 98 °C, 30s at 57 °C, 1 min. at 72 °C, and a final step of 10 min. at 72 °C. Barcoded samples were then sequenced on the HiSeq2000 in 2 × 100-bp paired-end mode.