Trypsin 0.05 edta 0 02

For triple-transfection studies, the human embryonic kidney cell line HEK293 (Stratagene, La Jolla, CA, USA) was used and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin–streptomycin (Biochrom, Berlin, Germany) at 37 °C and 5% CO₂ in a humidified incubator. Passaging of the cells was carried out with Trypsin (0.05%)-EDTA (0.02%) (Biochrom, Berlin, Germany) treatment followed by Trypsin-EDTA inactivation with Solution A (7.25 g HEPES; 1.8 g glucose; 0.22 g NaCl; 0.27 g Na₂HPO₄; 1 mL phenol red; 900 mL H₂O; pH 7.4) supplemented with 10% FBS. Cells were centrifuged at 350× g for 5 min and seeded in fresh tissue culture 6-well plates or 60 mm plates.
Transient transfections of screening plasmids were performed the next day using the jetPEI reagent (Polyplus-transfection SA, Illkirch, France) according to the manufacturer’s protocol. The transfected DNA amounts varied in the different experiments: Section 2.2: 0.5 µg KRT14-MG + 0.5 µg RTM + 3 µg asRNA were transfected into HEK293 cells cultivated in 6-well plates; Section 2.3: 0.5 µg KRT14-MG + 0.5 µg RTM + 70 nM ASO were transfected into HEK293 cells cultivated in 6-well plates.

For co-transfection experiments the human embryonic kidney cell line HEK293AD (Stratagene, La Jolla, CA, USA) was grown in DMEM supplemented with 10% fetal calf serum (FCS) and 100 U/mL penicillin/100 μg/mL streptomycin (Biochrom, Berlin, Germany) at 37 °C and 5% CO₂ in a humidified incubator. Trypsin (0.05%)-EDTA (0.02%) (Biochrom, Berlin, Germany) was used for detachment of cells, and cells were pelleted by centrifugation at 350× g for 5 min. For transient transfection of DNA the jetPEI reagent (Polyplus-transfection SA, Illkirch, France) was used according to the manufacturer’s instructions. The transfected DNA amounts varied in the different experiments: Section 2.1: 1.5 μg target molecule + 1.5 μg RTM were transfected into HEK293 cells cultivated in a 6 well plate; Section 2.2 and Section 2.4: 3 μg COL7A1-MG + 3 μg dRTM were co-transfected into HEK293 cells and 5 μg of dRTM were transfected into the stably COL7A1-MG-expressing cell line cultivated in 60 mm plates. The RDEB patient keratinocytes carry a homozygous mutation (6527insC) in exon 80 of COL7A1 (RDEB-TA4) [26 (link)]. This cell line was cultivated in SFM medium (Life technologies, Carlsbad, CA, USA) including the provided supplements and 100 U/mL penicillin/100 μg/mL streptomycin (Biochrom) at 37 °C and 5% CO₂ in a humidified incubator.