Riboshredder

To remove residual DNA from total RNAs, 10 μg of RNA sample in a 50 μL reaction volume was treated with TURBO DNase (4 U) for 25 min, followed by its inactivation for 5 min using TURBO DNA-free Kit (Thermo Fisher Scientific). Total RNA was recovered (45 μL) by centrifuge at 10,000g for 2 min and the total RNA (1 μg) was converted to cDNA using SuperScript III First-Strand Synthesis system (Invitrogen) with random hexamer. When required, 5 pmol of gene specific primer was used for the gene specific cDNA synthesis. Reverse transcription was carried out at 50 °C for 1 h and terminated at 85 °C for 10 min. The residual RNA was removed using 2 μL of an enzyme mixture containing RNase H (2.5 U, New England Biolab) and Riboshredder (0.5 U, Epicentre) for each reaction. Approximately 10% of the reaction was used as the template for PCR amplification using GoTaq Green Master Mix (Promega) and the primer pairs listed in Supplementary Table 5. Cycling conditions were: 95 °C/3 min; 30 cycles of 94 °C/25 sec, 58 °C/25 sec, 72 °C/60 sec and a final 72 °C/5 min. The PCR products were separated by 2% agarose gel electrophoresis; DNA bands were eluted and cloned into pJET1.2 vector (Thermo Fisher Scientific) as recommended by the manufacturer’s instructions and the inserts were sequenced using the pJET1.2 reverse primer.