Zeocin selection reagent

As the wild type, ecotype Col-0 was used. Isolation of T-DNA knock out mutant rpt5a-4 (SALK_046321) and rpt2a-2 (SALK 005596) is described previously^{14 (link)}.
Sterilized seeds were germinated and grown on MGRL medium^{30 (link)} supplemented with 1% w/v sucrose (BioUltra grade, Sigma-Aldrich, St. Louis, MO, USA), solidified with 1% w/v agar purified (Nacalai Tesque, Kyoto, Japan). For zeocin treatment, Zeocin Selection Reagent (Thermo Fisher Scientific, Yokohama, Japan) was added at the indicated concentrations. Medium plates were horizontally placed in a growth chamber (22 °C 16-h light/8-h dark cycle).

Unless stated otherwise, 1.2 μg of the donor vectors along with 0.8 μg of the corresponding integrase-expressing plasmids, pCAG-NLS-HA-Bxb1 (Addgene #51271) (Hermann et al., 2014 (link)) and pCAG-φC31 (Addgene #62658) (Ohtsuka et al., 2015 (link)), were transfected by lipofection into AAVS1-Bxb1, AAVS1-φC31 and AAVS1-Dual hiPSC lines. For comparing the integration efficiency of donor vectors of different sizes, 35.76 fmol of each vector was transfected.
To integrate the modified RP11-10L20 BAC construct into the AAVS1-Bxb1 hiPSCs, Bxb1-BAC donor was co-electroporated with pCAG-NLS-HA-Bxb1. For both approaches, ∼3 days after transfection the cells were harvested and passaged so that they were ∼5% confluent the following day. To enrich for integrated hiPSCs, either blasticidin S hydrochloride (2 μg/mL, Sigma) or zeocin selection reagent (15 μg/mL, ThermoFisher) were added to the culture medium for a period of 12 and 5 days, respectively. Donor vector integration was confirmed via a PCR screening strategy to detect the formation of the two new recombination sites, attR and attL.