ZF_Infect_mut_map-sc-4474
ZF_Infect_mut_map
Identification of ENU-induced mutations in individual zebrafish (Danio rerio) covering all GRCz10 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.  Adult male zebrafish were mutagenised using ENU, G0 mutagenised individuals were outcrossed to create large F1 mutagenised libraries. Embryos were infected at 48 hpf through caudal vein injection and were phenotyped at either 5 dpi or 7 dpi. DNA was isolated via fin biopsy from F1, F2 or further subsequent generations or from pooled or single infected embryos. Fin biopsies were placed in 400 ?l of 100 ?g/ml proteinase K, embryos in 100ul for 10 h at 55°C, followed by 15 min at 85°C to heat inactivate the proteinase K. DNA was precipitated by adding 400 ?l of isopropanol and centrifuging for 30 min at 4000 rpm and 4°C. DNA pellets were washed twice with 400 ?l of 70% ethanol followed by centrifugation at 4000 rpm for 5 min, and resuspended in ddH20. DNA from each individual (1-2 ?g) was sheared and used to construct 150-200 bp insert Illumina libraries according to the manufacturers standard protocols.  This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Zebrafish exome pulldown for the identification of ENU induced mutations linked to phenotypes of infection.