ena-STUDY-INRA-15-01-2020-12:41:40:077-1963
Definition using LAM-PCR of the insertion profile of IFP2 transposons containing a NeoR using the wild type CRD-less PB transposase (1-558)
Integration assay. Assays were monitored as described (doi: 10.1371/journal.pone.0082559). Briefly, each sample of 100,000 cells in a well of a 24-well plates of plaque assays was co-transfected with 400 ng DNA plasmid and with equal amounts of donor of NeoR cassette included or not within a transposon and transposase sources (1:1 ratio). Two days post-transfection, each cell sample was transferred in a cell culture dish (100 mm diameter) and selected with a culture medium containing 800 µg/mL G418 sulfate (Eurobio Scientific, Les Ulis) for 15 days. After two washing with 1X saline phosphate buffer, about 2000 cell slones ere harvested for genomic DNA preparation using the DNeasy kit (Qiagen, Hilden, Germany). Linear amplification-mediated PCR (LAM-PCR) were performed to amplify the vector-genomic DNA junctions of piggybac and Tcr-pble vectors as described (doi: 10.1007/978-1-61779-603-6_15). All PCR were done using the high fidelity Q5 DNA Polymerase (New England Biolabs, Ipswich, MA). Final PCR products were purified, quantified and gathered in equimolar DNA amounts for each transposon vector (4 populations of LAM-PCR products) before to be used to make Illumina libraries using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® and NEBNext Multiplex Oligos for Illumina (New England Biolabs, Ipswich, MA). Fragment size selection, library quality control and Illumina sequencing (MiSeq 250 nucleotides, TruSeq SBS Kit v3) were achieved at the Plateforme de Séquençage Haut Débit I2BC (Gif-sur-Yvette, France). DNA quantities were monitored at various steps in the procedure with the Qubit® dsDNA (Molecular Probes, Eugene, USA).
Integration assay. Assays were monitored as described (doi: 10.1371/journal.pone.0082559). Briefly, each sample of 100,000 cells in a well of a 24-well plates of plaque assays was co-transfected with 400 ng DNA plasmid and with equal amounts of donor of NeoR cassette included or not within a transposon and transposase sources (1:1 ratio). Two days post-transfection, each cell sample was transferred in a cell culture dish (100 mm diameter) and selected with a culture medium containing 800 µg/mL G418 sulfate (Eurobio Scientific, Les Ulis) for 15 days. After two washing with 1X saline phosphate buffer, about 2000 cell slones ere harvested for genomic DNA preparation using the DNeasy kit (Qiagen, Hilden, Germany). Linear amplification-mediated PCR (LAM-PCR) were performed to amplify the vector-genomic DNA junctions of piggybac and Tcr-pble vectors as described (doi: 10.1007/978-1-61779-603-6_15). All PCR were done using the high fidelity Q5 DNA Polymerase (New England Biolabs, Ipswich, MA). Final PCR products were purified, quantified and gathered in equimolar DNA amounts for each transposon vector (4 populations of LAM-PCR products) before to be used to make Illumina libraries using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® and NEBNext Multiplex Oligos for Illumina (New England Biolabs, Ipswich, MA). Fragment size selection, library quality control and Illumina sequencing (MiSeq 250 nucleotides, TruSeq SBS Kit v3) were achieved at the Plateforme de Séquençage Haut Débit I2BC (Gif-sur-Yvette, France). DNA quantities were monitored at various steps in the procedure with the Qubit® dsDNA (Molecular Probes, Eugene, USA).