Definition using LAM-PCR of the insertion profile of IFP2 transposons containing a NeoR using the murine PGBD5 isoform, Mm523.

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 60,000 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).

整合实验（Integration assay）。本实验的监测方法参照已发表文献（doi: 10.1371/journal.pone.0082559）。简言之，取24孔噬斑培养板每孔中的100,000个细胞样本，与400 ng DNA质粒共转染，同时按1:1比例加入等量的供体NeoR盒（NeoR cassette，是否包含于转座子（transposon）中）以及转座酶（transposase）来源试剂。转染两天后，将每一份细胞样本转移至直径100 mm的细胞培养皿中，使用含800 μg/mL 硫酸G418（Eurobio Scientific公司，吕利，法国）的培养基筛选15天。用1×磷酸盐缓冲盐水洗涤两次后，收集约60,000个细胞克隆，使用DNeasy试剂盒（Qiagen公司，希尔德，德国）提取基因组DNA。采用线性扩增介导的PCR（Linear amplification-mediated PCR，LAM-PCR）扩增piggyBac与Tcr-pble载体的载体-基因组DNA连接片段，方法参照已发表文献（doi: 10.1007/978-1-61779-603-6_15）。所有PCR反应均使用高保真Q5 DNA聚合酶（New England Biolabs公司，伊普斯维奇，马萨诸塞州，美国）进行。将最终的PCR产物进行纯化、定量，并将每种转座子载体的LAM-PCR产物（共4组）按等摩尔浓度混合，随后使用NEBNext® Ultra™ II DNA文库制备试剂盒（适配Illumina®平台）以及NEBNext多重寡核苷酸接头（New England Biolabs公司，伊普斯维奇，马萨诸塞州，美国）构建Illumina测序文库。片段大小筛选、文库质量控制以及Illumina测序（使用MiSeq平台，读长250 nt，配套TruSeq SBS Kit v3试剂盒）均在I2BC高通量测序平台（法国吉夫于维特）完成。实验过程中各步骤的DNA浓度均使用Qubit® 双链DNA检测试剂盒（Molecular Probes公司，尤金，美国）进行定量监测。