CCF_3255
WXS of meningioma tissue
DNA from flash-frozen tumour 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 vendors protocol v2.0.1. The SureSelect Human All Exon Kit targets regions of 50Mb in total size, which is approximately 1.7% of the human genome. In brief, 3g genomic DNA was sheared with a Covaris S2 to a mean size of 150bp. Five hundred nanograms of library DNA was hybridized for 24h at 65C with the SureSelect baits. 15 ng final exome-enriched library (without barcode) was used as a template in a 50-l 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 2min at 98C was followed by four cycles of 30s at 98C, 30s at 57C, 1min. at 72C, and a final step of 10min. at 72C. Barcoded samples were then sequenced on the Hiseq2000 in 2100-bp paired-end mode.
CCF_3255
SUB6449015
CCF_3255