2P-Seq of rice: Pooled_N-starved
2P-Seq library construction procedure was followed described previously (Spies et al., 2013). Briefly, poly-A-tailed RNA was selected using oligo(dT)25 beads (NEB) and eluted directly into 25 ul of RNase T1 sequence buffer. RNase digestion was performed using 0.5 U RNase T1 (Ambion) then digested RNAs were precipitated using glycogen (20 ug/ul). Partially digested RNA was reverse-transcribed with SuperScript III Reverse Transcriptase (Invitrogen), and synthesized first-stranded cDNA was purified on a denaturing polyacrylamide gel, then circularized using CircLigase II (EpiBio). CircLigase II was heat-inactivated for 10 min at 80°C. Circularized cDNA was amplified by PCR for 12–15 cycles using barcoded PCR primers, then gel-purified.