Wizard genomic dna purification

DNA was extracted from organs or blood (Wizard Genomic DNA Purification, Promega). MCMV coordinates 111218–111461 were amplified (LightCycler 480 SYBR green, Roche Diagnostics) and converted to genome copies by comparison with plasmid DNA amplified in parallel. Cellular DNA was quantified in the same samples by amplification of a β-actin gene segment. Viral DNA loads were normalized by cellular DNA loads.

We extracted purified DNA from the isolates using Wizard® Genomic DNA Purification (Promega) kits and QIAGEN© DNEasy Blood & Tissue Kits according to the manufacturer’s instructions. Whole-genome sequencing of E. coli isolates was carried out at the University of Minnesota using Illumina MiSeq with Nextera XT libraries. Raw reads were quality-trimmed and adapter-trimmed using trimmomatic^{40 (link)} and assembled using SPAdes (Bankevich et al. 2012). Antibiotic resistance genes (ARGs) were detected using ABRicate (version 0.813) and a curated version of the ResFinder database (Zankari et al. 2012). We also performed in silico multilocus sequence typing (MLST) based on seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA), an additional eight housekeeping genes (dinB, icdA, pabB, polB, putP, trpA, trpB, and uidA), and core genome (cgMLST) using MLST 2.0^{41 (link)} and cgMLSTFinder 1.1^{42 (link)}. Detailed methods are previously described^{43 (link)}.

Transgenic chickens were prepared using surrogate shell methods [29 (link)]; all of the White Leghorn donor eggs used in the experiments were from newly fertilized eggs obtained from the North Agricultural Technology company. The subgerminal cavity beneath the blastoderm at stage X was microinjected with 1–2 μl of viral suspension and then placed in the first recipient eggshells and incubated at 37°C at 50%-60% humidity for three days. The embryos were then transferred to a second series of surrogate shells and incubated under the same conditions until hatched.
To identify the transgene in chickens, DNA was extracted from heart, liver, spleen, lung, kidney and muscle of the chicken embryos using a genomic DNA purification kit (Promega, Wizard Genomic DNA Purification) and PCR was performed. To determine the efficiency of lentiviral infection, 15 three-month-old G₀ hens were randomly slaughtered, and the transgene was identified in the heart, liver, spleen, lung, kidneys and ovaries of these animals.
The amount of DNA used for PCR amplification in G₀ chimeric chickens was 1 μg. Because the primers used for screening the transgenic chickens did not include the His-tag sequences, the transgenic chickens with the HNP4 gene or HNP4-His gene could be detected by the same primers.

DNA samples were extracted from young leaves (2- to 3-wk-old) taken from each line, using Wizard Genomic DNA purification (Promega) following the manufacturer’s protocol. DNA samples were genotyped using an Illumina 9K SNP chip with 8632 SNPs (Cavanagh et al. 2013 ). For a given marker, the genotype for the ith line was coded as the number of copies of a designated marker-specific allele carried by the ith line (absences equal to zero, and presents equal to one). SNP markers with unexpected genotype AB (heterozygous) were recoded as either AA or BB, based on the graphical interface visualization tool of GenomeStudio (Illumina) software. SNP markers that did not show clear clustering patterns were excluded. In addition, 66 simple sequence repeat (SSR) markers were screened. After filtering the markers for 0.05 minor allele frequency (MAF), and deleting markers with more than 10% of no calls, the final set of SNPs was 1635 SNPs.