Antisense Transfection of HOTAIR in RAW 264.7 Cells

For the antisense transfection, RAW 264.7 cells were grown up to 60% confluency in 60 mm culture plates and transfected with HOTAIR-antisense (HOTAIR-AS) and scramble antisense (Scr-AS, no homology to HOTAIR) oligonucleotides^{56 (link),57 (link)} independently (Table 1) using GenMute siRNA and DNA transfection reagent (SL100568, SignaGen Laboratories) according to the manufacturer’s protocol. A cocktail of transfection reagent and antisense oligonucleotides was made prior to transfection. Initially, 12 μL (12 μg) of GenMute reagent was mixed with 300 μL DMEM (minus FBS and antibiotics) in an eppendorf tube. Antisense oligonucleotides were mixed with 100 μL DMEM (without supplements) in a separate eppendorf. Then the diluted antisense solution was mixed with diluted GenMute reagents and allowed to stand for 30 min in the dark. In the intervening time, cells were washed twice with supplement-free DMEM and then 1.7 mL of supplement-free DMEM was added to each cell culture plate. Finally, antisense transfection reagents cocktail was applied to the cell plates, mixed gently and incubated for 48 h. Cells were then stimulated with LPS (1 μg/mL) for specified time period and then harvested for RNA extraction.