Dharmacon on target plus smart pool sirna

Cells were seeded at 50,000 cells per well in a 6-well plate and incubated for 24 h at 37 °C. Thereafter, they were transfected with Dharmacon ON-TARGET plus SMART pool siRNA (Horizon Discovery, Cambridge, UK) targeting INO80, and the negative control, Dharmacon ON-TARGET plus non-targeting pool (Horizon Discovery) using Lipofectamine™ RNAiMAX Reagent (Thermo Fisher Scientific) and Opti-MEM^® (Thermo Fisher Scientific). The cells were incubated at 37 °C and harvested after 48 h.

The circadian rhythm was synchronized via stimulation of 0.1 µM Dex (Wako, Osaka, Japan) for 2 h or 10 µM Frk (Wako, Osaka, Japan) for 1 h. After each stimulation, the medium was replaced with a standard medium (referred as 0 h), and RNA extraction was performed every 4 h. The initial induction of PER1 was examined by adding 0.1 µM Dex or 10 µM Frk. RNA extraction was performed every 30 min, while the time of addition was 0 min. The simulated body temperature rhythm was achieved by using a 33 °C condition for 12 h and a 37 °C condition for 12 h, for a total of 5 days, and then the cells were cultured for another 24 h at 37 °C. RNA extraction was performed every 4 h from day 5 to day 6. Gene knockdown was performed using Dharmacon™ ON-TARGETplus SMARTpool™ siRNA (Horizon Discovery, Lafayette, CO, USA), which consists of a mixture of four siRNAs for same target gene. Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) was used to add a final concentration of 20 nM siRNA. siRNA was added on day 4 of the temperature rhythm, and removed 24 h later. Hypoxic treatment was performed by culturing cells under a 1% O₂ condition for the indicated periods in a multi-gas incubator (APM-30DR; ASTEC, Fukuoka, Japan).