barcoded oxford nanopore strategy  (Oxford Nanopore)

Journal: PLoS ONE

Article Title: A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station

doi: 10.1371/journal.pone.0253403

Figure Lengend Snippet: A. Map of the pVG1 vector . This vector contains CRISPR machinery: Cas9, guide RNAs targeting ADE2 , a repair template that introduces two stop codons and an EcoRI site into the ADE2 gene, and the URA3 gene for positive selection. B. ADE2 mutant colonies are easily distinguished from those bearing the wild type ADE2 sequence. ADE2 is not essential for survival, but S . cerevisiae with mutations in this gene turn red due to the buildup of purine precursors in the vacuole . Wild type S . cerevisiae colonies are white. C. Adaptation of S . cerevisiae transformation and CRISPR/Cas9 genome editing protocols for use onboard the ISS. Prior to launch, cells were grown in liquid culture on Earth, pelleted by centrifugation, and frozen in glycerol at -80°C for transport to the ISS. Step 1, transformation: transformation mixture and pVG1 vector were added to thawed cells. The miniPCR thermal cycler was used as a heat block to induce transformation. Following transformation, cells were plated on synthetic defined agar lacking uracil (SDA-URA) and grown at room temperature for six days when the phenotype of the colonies was assessed. Step 2, DNA extraction: A pipette tip was used to transfer a small number of cells from four red and four white colonies to the DNA extraction buffer. Cells were heated in the miniPCR thermal cycler to 95°C to extract the DNA. Step 3, PCR and barcoding: DNA extract was directly added to PCR reagents. PCR was performed to amplify the 5’ end of the ADE2 gene. Sequencing barcodes were added at this step. Step 4, sample pooling and magnetic bead clean up: PCR product was pooled and purified during a magnetic bead cleanup step. Step 5, nanopore sequencing: Purified PCR product was sequenced by nanopore sequencing. Data was downlinked to the ground where sequences were assessed.

Article Snippet: For a more detailed explanation of Oxford Nanopore Technologies’ barcoding strategies, see Matsuo et al., 2021 [ ].

Techniques: Plasmid Preparation, CRISPR, Selection, Mutagenesis, Sequencing, Transformation Assay, Centrifugation, Blocking Assay, DNA Extraction, Transferring, Purification, Nanopore Sequencing