Mission shrna control vector

HCT116 or MCF-7 cells were infected with MISSION shRNA control vector and specific p27 or PCAF shRNA (Sigma Aldrich) with the following sequences:
p27: 5΄-CCGGGTAGGATAAGTGAAATGGATACTCGAGTATCCATTTCACTTATCCTACTTTTTG-3΄
PCAF:5΄-CCGGGCAGACTTACAGCGAGTCTTTCTCGAGAAAGACTCGCTGTAAGTCTGCTTTT-3΄
The protocol for viral particles production and cell infections has been described elsewhere (34 (link)). Twenty four hours after infection, cells were selected with 2 mg/ml of Puromycin (Sigma Aldrich) for five days. shRNA-mediated down-regulation was tested by WB with specific antibodies.

Vectors (pcDNA3.1) containing LINK-A lncRNA short hairpin RNA (shRNA 5′-AAAAGCTTCTCTCACCCTTCAAATTGGATCCAATTTGAAGGGTGAGAGAAGC-3′) were designed and synthesized by Shanghai GeneChem Co., Ltd. (Shanghai, China). MISSION shRNA Control Vector was purchased from Sigma-Aldrich (cat. no. SHC001; Merck KGaA). LINK-A (NCBI ID, NR_015407.1) lncRNA overexpression vectors (pcDNA3.1) and empty vectors (pcDNA3.1) were provided by Shanghai Sangong Pharmaceutical Co., Ltd. (Shanghai, China). Lipofectamine^® 2000 reagent (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to transfect 15 nM vectors into 5×10⁵ MG-63 and U2OS cells. Transfections were performed at 37°C for 6 h. Non-transfected cells were used as control cells. Cells transfected with empty vectors were used as negative transfection control cells. Transfection efficiency was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cells were harvested 24 h post-transfection prior to subsequent experiments.