Cell line buffer sf

siRNA nucleofection was performed with the Amaxa 96-well shuttle system (Lonza)^{35 (link)}. Pre-confluent human keratinocyte cultures were disaggregated and re-suspended in cell line buffer SF (Lonza). For each 20 μl transfection (program FF-113) reaction, 2 × 10⁵ cells were mixed with 1 μM siRNA duplexes as described previously^{33 (link),35 (link)}. Transfected cells were allowed to recover at ambient temperature for 10 min and were subsequently re-plated onto rat-tail type I collagen (20 µg ml⁻¹ in PBS, BD Biosciences)-coated cell culture well plates (Falcon) and grown in KSFM for 24 h before being used for downstream assays^{35 (link)}. 27mer siRNA oligo duplexes (SR509346, Origene) were used for gene knockdown of human DLL1 (the sequences of siRNA oligos can be found in Supplementary Table 4). Non-targeting control siRNAs were from Ambion (AM4611 and AM4637).

siRNA nucleofection was performed with the Amaxa 96-well shuttle system (Lonza). Pre-confluent NHK cultures were disaggregated and resuspended in cell line buffer SF (Lonza). For each 20 μl transfection (program FF-113) reaction, 2 × 10⁵ cells were mixed with 1 μM siRNA duplexes as described previously15 (link). Transfected cells were allowed to recover at ambient temperature for 10 min and were subsequently re-plated into rat-tail type I collagen (20 μg ml⁻¹ in PBS, BD Biosciences)-coated cell culture well plates (Falcon) and, unless stated otherwise, grown in KSFM medium for 24 h before being used for downstream assays. SMARTpool ON-TARGET plus siRNAs (GE Dharmacon) were used for gene knockdown. Each SMARTpool was a mix of four sets of RNAi oligos (the sequences of siRNA oligos can be found in Supplementary Table 3). Non-targeting control siRNAs were from Ambion (AM4611 and AM4637). SCC13 cells (5 × 10⁵) were reverse transfected with siRNAs (25 pmol) in six-well dishes (Falcon) using Lipofectamine RNAiMAX reagent in combination with Opti-MEM medium (Life Technologies) according to the manufacturer's instructions.