Genomic DNA from neor-positive HeLa cells was purified using the Genelute
Mammalian Genomic DNA Kit (Sigma), and the PureLink Genomic DNA Mini Kit
(Invitrogen) was used for CD19 CAR T cells. The average number of
neomycin-resistance transgene insertions was measured by digital droplet PCR as
follows: 200 ng of genomic DNA (gDNA) was digested with 20 units of DpnI
restriction enzyme (NEB) in 30 μl final reaction volume overnight to
eliminate non-integrated transposon plasmid DNA present in the samples. Next,
the DNA was fragmented with CviQI (NEB) for 4 h at 25 °C. The samples
were subjected to PCR amplification using Taqman probes and primers specific for
the right end of the transposon and for the single-copy human RPP30 gene to
measure the genome count. The PCR reactions were performed in 20 μl final
volume with 10 ng of fragmented gDNA using the ddPCR Supermix for Probes (No
dUTP) master mix (Bio-rad) with 11 pmol primers and 50 pmol of TaqMan probes
(for sequences of primers and probes seeSupplementary Table 2 ). The PCR droplets were generated in
the QX100 device (Bio-rad). The program was 95 °C 10 min; 40 cycles of 94
°C 30 s, 50 °C 30 s, 60 °C 1 min; 98 °C 10 min.
After thermal cycling, the fluorescent droplets were counted in the QX100
Droplet Reader (Bio-rad), and genomic copy numbers were calculated with the
Quanta Soft program (Bio-Rad).
Mammalian Genomic DNA Kit (Sigma), and the PureLink Genomic DNA Mini Kit
(Invitrogen) was used for CD19 CAR T cells. The average number of
neomycin-resistance transgene insertions was measured by digital droplet PCR as
follows: 200 ng of genomic DNA (gDNA) was digested with 20 units of DpnI
restriction enzyme (NEB) in 30 μl final reaction volume overnight to
eliminate non-integrated transposon plasmid DNA present in the samples. Next,
the DNA was fragmented with CviQI (NEB) for 4 h at 25 °C. The samples
were subjected to PCR amplification using Taqman probes and primers specific for
the right end of the transposon and for the single-copy human RPP30 gene to
measure the genome count. The PCR reactions were performed in 20 μl final
volume with 10 ng of fragmented gDNA using the ddPCR Supermix for Probes (No
dUTP) master mix (Bio-rad) with 11 pmol primers and 50 pmol of TaqMan probes
(for sequences of primers and probes see
the QX100 device (Bio-rad). The program was 95 °C 10 min; 40 cycles of 94
°C 30 s, 50 °C 30 s, 60 °C 1 min; 98 °C 10 min.
After thermal cycling, the fluorescent droplets were counted in the QX100
Droplet Reader (Bio-rad), and genomic copy numbers were calculated with the
Quanta Soft program (Bio-Rad).