Nonspecific control sirna

The two siRNAs specific for SETDB1 named S5 and S9 and a nonspecific control siRNA were purchased from Invitrogen (Invitrogen, Shanghai, China). The specific siRNA sequences for SETDB1 used in this study were as follows:
S9 sense sequence: 5’-CGUGACUUCAUAGAGGAGU-3’; antisense sequence: 5’-ACUCCUCUAUGAAGUCACG-3;
S5 sense sequence: 5’-GAUCUAUCGAGGCUCUACA-3’; antisense sequence: 5’-UGUAGAGCCUCGAUAGAUC-3’.
An evaluation of the efficiency of the siRNA knockdown experiments was performed. We compared the SETDB1 expression in normal 22RV1 cells cultured for 72 h with S5 and S9 siRNA 72 h post-infected 22RV1 cells and siRNA control post-infected 22RV1 cells. The transfection method and gene expression assay method are the same as those described above.

We designed the siRNA to target myeloid differentiation primary response gene 88 (MyD88). The MyD88 siRNA and a non-specific control siRNA were chemically synthesized by Invitrogen (China). The siRNA sequences used were as follows: MyD88, 5'-AUGCCUGAGCAUUUUGAUGTT-3' (sense) and 5'-CAUCAAAAUGCUCAGGCAUTT-3' (antisense); and non-specific control siRNA, 5'-CUGCCCCAGCGAUAUCCAGTT-3' (sense) and 5'-CUGGAUAUCGCUGGGGCAGTT-3' (antisense). PAMs were transfected with 10 nM siRNA using Lipofectamine 2000 (Invitrogen, China) for 24 h and were then inoculated with PCV2 virus after washing twice with PBS buffer. The siRNA dose was optimized in pilot experiments, and no appreciable cellular toxicity was observed.

RAW264.7 cells were treated with siRNA specific to Dusp2 or nonspecific control siRNA (Life Technologies) (Table 1). Cells were transiently transfected in Opti-MEM I medium using Lipofectamine RNAiMAX (Life Technologies). The efficiency of silencing was assessed with PCR 48 h after transfection.