Complemented palindrome small RNAs first discovered from SARS coronavirus

Chang Liu; Ze Chen; Wenyuan Shen; Deshui Yu; Siyu Li; Yue Hu; Haishuo Ji; Wenjun Bu; Qingsong Wang; Shan Gao

Based on the shRNA design protocol [1], the sequences of SARS-CoV-cpsR-22, its 16-nt, 18-nt, 19-nt 218 20-nt segments ( Figure 2B ) and their control "CGTACGCGGAATACTTCGA" were selected to use as 219 target sequences for pSIREN-RetroQ vector construction (Clontech, USA), respectively. PC-9 cells were 220 divided into six groups named 22, 16, 18, 19, 20 and control for transfection using plasmids containing 221 20 -nt segments and control sequences. Each group had three 222 replicate samples for plasmid transfection and cell apoptosis measurement. Each sample was processed 223 following the same procedure described below. At 12 h prior to transfection, the PC-9 cells were washed 224 with PBS and trypsinized. Gbico RPMI-1640 medium was added into the cells, which were then centrifuged 225 at 1000 rpm for 10 min at 4°C to remove the supernatant. Gbico RPMI-1640 medium (Thermo Fisher 226 Scientific, USA) containing 10% fetal bovine serum was added to adjust the solvent to reach a volume of 2 227 μ L and contain 2 ╳ 10 5 cells. These cells were seeded in one well of a 6-well plate for plasmid transfection.