We transfected 1 × 10^6 human fibroblasts with CRISPR/Cas9 activators, as previously described [23 (link)]. Briefly, we detached the fibroblasts as single cells and washed them with PBS. Using the Neon transfection system (ThermoFisher; 1650 V, 10 ms, and 3 pulses), we electroporated 6 μg of plasmid mixture, (2 μg of dCas9 activator plasmid and 4 μg of guide plasmids) into the cells in a 100 μl transfection tip. Treated fibroblasts were plated on Matrigel -coated plates (Corning) in DMEM;10%FBS (ThermoFisher), later (d4) changed to a 1:1 mixture of DMEM;10%FBS and hES-medium (Gibco) supplemented with sodium butyrate (0.25 mM; Sigma).
iPSC colonies were picked manually (d15) and plated on Matrigel-coated wells in E8 medium. Media were changed every other day and cells were expanded up to passage 10. Thereafter, we used PCR targeting EBNA1 (Fw: 5’-ATCGTCAAAGCTGCACACAG −3′; Rv: 5’-CCCAGGAGTCCCAGTAGTCA −3′, Sigma) and OriP (Fw: 5’-TTCCACGAGGGTAGTGAACC -3′; Rv: 5’-TCGGGGGTGTTAGAGACAAC -3′, Sigma) to confirmed that the reprogramming plasmids had not integrated into the genome [24 (link)].
iPSC colonies were picked manually (d15) and plated on Matrigel-coated wells in E8 medium. Media were changed every other day and cells were expanded up to passage 10. Thereafter, we used PCR targeting EBNA1 (Fw: 5’-ATCGTCAAAGCTGCACACAG −3′; Rv: 5’-CCCAGGAGTCCCAGTAGTCA −3′, Sigma) and OriP (Fw: 5’-TTCCACGAGGGTAGTGAACC -3′; Rv: 5’-TCGGGGGTGTTAGAGACAAC -3′, Sigma) to confirmed that the reprogramming plasmids had not integrated into the genome [24 (link)].
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