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. 2016 Jan 8;7:10286. doi: 10.1038/ncomms10286

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(a) Schematic of the human CADPS2 allele of the hiPS-SB4 line harbouring insertion L1-dn13. A CADPS2 transcript including exons 7, 8, 27 and 28 is presented. Binding sites of the TaqMan primer/probe combination spanning the exon27/exon28 junction on CADPS2 cDNA used for qRT–PCR analysis are shown (red arrows and line). (b) Relative CADPS2 mRNA levels in early (p16) and late passage (p50) hiPS-SB4 cells were assessed by qRT–PCR. HES-3 and hFF-iPS4 cells served as positive controls. qRT–PCR results were normalized to 18S rRNA using CADPS2 expression in parental HFF-1 cells as control. Bars, arithmetic means±s.e.m. of technical triplicates. (c) Structure of the L1-dn13 integration site in the CADPS2 gene in hiPS-SB4 subclones. hiPS-SB4_D differs from hiPS-SB4_B by the presence of the L1-dn13 de novo insertion in CADPS2 intron 7. Binding sites of L1-dn13-specific validation PCR primers OP1, ISP1 and ISP2 and expected lengths of the resulting PCR products are indicated. Black diamonds, TSDs. (d) Genotyping PCR validating the L1-dn13 presence in subclone hiPS-SB4_D and its absence from hiPS-SB4_B in gDNAs isolated from HFF-1, hiPS_SB4_B, and hiPS_SB4_D cells and from the original mixed population of the hiPS-SB4 culture (hiPS-SB4(Mix)). Primer combinations used are indicated in blue; H₂O.ISP1/ISP2 and H₂O.ISP1/OP1, negative control PCRs using H₂O instead of gDNA; 100-bp ladder, size marker. (e) qPCR analyses confirming absence of L1-dn13 from hiPS-SB4_B and HFF-1 cells, and its presence in hiPS-SB4_D cells and the hiPS-SB4 culture. gDNAs from HFF-1 cells and from hiPS-SB4(Mix) cells served as negative and positive controls, respectively. For normalization, a primer/probe combination specific for the human RPP25 gene was used. ΔΔCt values measured the relative quantity of L1-dn13. Bars, arithmetic means±s.e.m. of technical triplicates. (f) Relative CADPS2 mRNA levels in hiPS-SB4_B, hiPS-SB4_D and hiPS-SB4(Mix) cells were determined by qRT–PCR using cytoplasmic RNA and primer/probe combinations spanning exon 27/exon 28 junction of CADPS2. Bars, arithmetic means±s.e.m. of technical triplicates.