ZF_kdm2aa_mut_whole_exome_enrichment. ZF_kdm2aa_mut_whole_exome_enrichment

Identification of point mutations in individual kdm2aa homozygous mutant zebrafish tumours covering all Zv9 annotated zebrafish protein-coding genes using the Agilent SureSelect whole exome enrichment reagent. Oligonucleotides (120mers) were designed at 2X tiling across the predicted exon coordinates and then manufactured as biotinylated RNA baits and blended into one tube ready for enrichment. DNA was isolated and extracted from kdm2aa homozygous mutant tumours, adjacent healthy looking tissue, and corresponding tissue from healthy wild-type or heterozygous siblings. Dissected tissue were placed in 400 ?l of 100 ?g/ml proteinase K for overnight at 55°C, followed by 30 min at 80°C to heat inactivate the proteinase K. DNA was precipitated by adding 400 ?l of isopropanol and centrifuging for 40 min at 4100 rpm t room temperature. DNA pellets were washed twice with 400 ?l of 70% ethanol followed by centrifugation at 4100 rpm for 25 and 10 min, and resuspended in ddH20.