1urq1 dnase stop solution

Total RNA was extracted using Sepasol RNAI Super (Nacalai Tesque, Inc.), as described by the manufacturer's instructions. The extracted total RNA samples were dissolved in TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) and stored at −80°C until use. The RNA samples were treated with RQ1 RNase-free DNase mixture (Promega Co., Madison, WI, USA; 1 µg total RNA, 1× DNase buffer, and 1 UDNase in 10 µl) on a programmable thermal controller (PTC-100; MJ Research, Waltham, MA, USA), programmed at 37°C for 30 min and then at 65°C for 10 min with 1URQ1 DNase Stop Solution (Promega Co.). The concentration of RNA in each sample was measured using Nano Drop Lite (Thermo Fisher Scientific., Waltham, MA, USA). The RNA samples were reverse-transcribed using Rever-Tra Ace (Toyobo Co., Ltd., Osaka, Japan) as per manufacturer's instructions. The reaction mixture (10 µl) comprised 0.5 µg total RNA, 1× reverse transcription buffer (Toyobo Co., Ltd.), 1 µM deoxyribonucleotide triphosphate (dNTP) mixture (Toyobo Co., Ltd.), 5 URNase inhibitor (Toyobo Co. Ltd.), 0.25 µg of oligo (dt) 20 (Toyobo Co., Ltd.), and 50U Rever Tra Ace. The reverse transcription was performed at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min using a programmable thermal controller. Finally, the cDNA samples were stored at −20°C until use.

Total RNA was extracted using Sepasol RNA I Super (Nacalai Tesque, Inc. Kyoto, Japan) per the manufacturer's instructions. The extracted total RNA samples were dissolved in TE butter (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and stored at −80°C until further use. The RNA samples were treated with 1URQ1 RNase-free DNase (Promega Co. Madison, WI, USA) in a 10 µl reaction mixture (1 µg total RNA, 1× DNase buffer and 1UDNase) on a programmable thermal controller (PTC-100; MJ Research, Waltham, MA, USA), programmed at 37°C for 30 min and then at 65°C for 10 min with 1URQ1 DNase Stop Solution (Promega Co.). The concentration of RNA in each sample was measured using a NanoDrop Lite (Thermo Fisher Scientific, Waltham, USA). The RNA samples were then reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan) according to the manufacturer's instructions. The reaction mixture (10 µl) consisted of 0.5 µg total RNA, 1× reverse transcription buffer (Toyobo Co. Ltd.), 1 µM deoxyribonucleotide triphosphate (dNTP) mixture (Toyobo Co. Ltd.), 5U RNase inhibitor (Toyobo Co. Ltd.), 0.25 µg oligo(dT) 20 (Toyobo Co. Ltd.), and 50 UReverTra Ace. Reverse transcription was performed at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min using a programmable thermal controller. The cDNA samples were stored at −20°C until use.