Sigenome human cd274 sirna smartpool

To decrease PD-L1 expression (KD), cells were transiently transfected with small interfering RNA (siRNA) directed against the human PD-L1 gene CD274. Dharmafect-1 (Thermo Scientific) was used as the transfection reagent according to the manufacturer’s instructions.
This siRNA was designed to reduce off-target effects by more than 90%. The siRNA consisted of a pool of 4 different siRNAs (the sequences are listed below in Table 1) so that elimination was guaranteed. Seed regions play a key role in causing off-target effects. To avoid these seed region caused off-target effects, special design filters were applied to eliminate frequent seed regions. Transfection was performed in 6-well plates (Corning, NY, USA). For reverse transfection, a cell suspension of 2 × 10⁵ cells was added to prepared transfection complexes. Transfection with 25 nM siRNA was carried out in DMEM growth medium without antibiotics for 72 h. The siGENOME Human CD274 siRNA SMARTpool (M-015836-01-0005, Horizon Discovery, Cambridge, UK) consisted of four siRNA target sequences. The ON-TARGETplus Non-Targeting Control Pool (D-001810-10-20, Horizon Discovery) was used as a control. The SiRNA sequences used for PD-L1 knockdown experiments are listed in Table 1. Three days after transfection, cells were relocated for optimal confluence and used for experiments.

To decrease PD‐L1 expression (KD), cells were transiently transfected with small interfering RNA (siRNA) directed against the human PD‐L1 gene CD274. Dharmafect‐1 (Thermo Scientific, Waltham, MA, USA) was used as transfection reagent according to the manufacturer's instructions. Transfection was performed in 6‐well plates (Corning, NY, USA). A cell suspension of 2 × 10⁵ cells was added to prepared transfection complexes. Transfection with 25 nm siRNA was carried out in DMEM growth medium without antibiotics for 72 h. The siGENOME Human CD274 siRNA SMARTpool (M‐015836‐01‐0005, Horizon Discovery, Cambridge, UK) consists of four siRNA target sequences. This method has already been described in [21 (link)]. The ON‐TARGETplus Non‐Targeting Control Pool (D‐001810‐10‐20, Horizon Discovery) was used as control. The siRNA sequences used for PD‐L1 KD experiments are listed in Table 1.