Figure 2

(A) Overview of GBT screening pipeline. Wild-type embryos at 1 cell were co-injected with RP plasmid and Tol2 transposase mRNA to create F0 founders. These F0 larvae were screened for non-mosaic RP expression, raised, and outcrossed for two generations. Then, mRFP⁺ F2 heterozygous larvae were 3-dimensionally imaged at 2 and 4 dpf and this imaging data were uploaded to zfishbook (http://www.zfishbook.org/). Sperm from four F2 males in over 1200 robust mRFP expressing lines were cryopreserved using the Zebrafish International Resource Center (ZIRC) standard protocol and stored at both ZIRC and Mayo Clinic Zebrafish Core Facility (MCZF). DNA and RNA isolated from these four F2 males with cryopreserved sperm was utilized to perform next-generation sequencing and to confirm RFP linkage of candidate lines by manual PCRs (iPCR, TAIL-PCR, 5’ RACE and 3’ RACE). Venn diagram illustrates current library of over 1,200 GBT lines with 204 GBT-confirmed lines out of 348 molecularly analyzed GBT-candidate lines. (B) Next generation sequencing based validation for GBT integration loci. Fin biopsies from four F2 males were utilized as DNA source for the validation process to identify GBT integration loci. Extracted genomic DNA was fragmented, pooled in 96-wells plate, and ligated with barcode linker to identify each single male with cryopreserved sperm. Linker-mediated (LM) PCR with the primers, R-ITR P1 and LP1 and nested PCR with the primers, R-ITR P2 and LP2 were conducted to perform Illumina sequencing the final PCR products. The integration events of individual sperm-cryopreserved male were mapped on zebrafish reference genome sequence with bioinformatics analysis. This figure was created with BioRender.com. The area proportional Venn diagram was produced using BioVenn (http://www.biovenn.nl/).