Sigenome human smartpool

Dharmacon siGENOME Human SMARTpool siRNAs for HUR1, RIG-I or a non-relevant target (NR) were transfected into THP-1 cells using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Protein expression was measured by western blot analysis 48 h post transfection.

Endothelial cells were transfected with 4 siRNAs for each target (siGENOME Human SMARTpool, Dharmacon Lafayette, Colorado, USA) with Lipofectamine 3000 (Invitrogen, Thermo Fisher Waltham, MA, USA) according to the manufacturer’s instructions. The siRNA sequences are as follows: NID1: GGGCGAACCUGCUAUGAUA, GAAGGUUUAUUAUCGAGAA, UAACCUGGAUCGAAUAGAA, and CCUUCAUAACUGCGACAUA; BGN: GGAGAACAGUGGCUUUGAA, UGAAUGAACUCCACCUAGA, CCAAAGAGAUCUCCCCUGA, and GAACAACGACAUCUCCGAG; HSPG2: GCGCUGCGAUGGUGACUUU, CAACACACACCACGAGCUA, GAGCUAUGUGAAUGCAAU, and ACGGUGGGAAGUUGCGAUA; CLU: GAUAAAGACUCUCAUAGAA, GAAAGAGGAUGCCCUAAAU, GGAAGUAAGUACGUCAAUA, and GUAGAAGUCUCCAGGAAGA; C1QTNF5: GCGAAUCCAUUGCCUCUUU, UGAACGAGCAGGGACAUUA, GGGCCAGCCUGCAGUUUGA, and UGACUACAUUGGCAUCUAU; CYR61: GGGCAGACCCUGUGAAUAU, GGCCAGAAAUGUAUUGUUC, GGUCAAAGUUACCGGGCAG, and GCAGCAAGACCAAGAAAUC; TIE1: GGGAAGCCUCCUACCCUUA, GAAGUUCUGUGCAAAUUGG, CAACAUGGCCUCAGAACUG, and UCGAAACUGUGACGAUGAA; VWF: GGACAGAUCAUGACACUGA, GGAAGACCCUGUGGACUUU, GAAGAGGCCUGCACUCAGU, and GGUCACAUCUUCACAUUCA. After 72 h, the transfection medium was changed, and a second transfection was performed. Twenty-four hours after the second siRNA transfection, the cells were used for experiments.

Dharmacon siGENOME Human SMARTpool custom siRNA library targeting human serine/threonine kinases was obtained from Dharmacon RNA Technologies (Thermo Scientific, Dharmacon RTF # H-004405). The library contains a total of 47 siRNA cocktails with each cocktail consisting of 4 siRNA sequences targeting a specific gene and resuspended at a concentration of 2μM in 96-well plates. The separate and individual siRNA pools that were used to validate the hits identified from the screen was also obtained from Dharmacon (siGENOME SMARTpool): MINK (M-004861–03–0005), MAP4K2 (M-003587–01–0005). The sequences of the respective scrambled siRNA obtained from Origene are: 5’-GAUUAAACGCAUGGCCUUU-3’, 5’-GUAAGAGCACAAGUCGUGG-3’, 5’-UCUAGAAGACUUUGGAAGA-3’ and 5’-GGCUAUAUUCUCUGUUGAC-3’. For the deconvolution of MINK siRNA SMARTpool, individual siRNAs were also obtained from Dharmacon (MQ-004861–03–0005).