title: Strand-specific RNA-Seq of rice: Root_-N_3d
design: For strand-specific RNA-Seq analysis, sequencing libraries were generated according to protocol previously described (Zhong et al., 2011). Briefly, poly-A-tailed RNA was selected from 5 ug of total RNA using oligo(dT)25 beads (NEB). RNA was fragmented by incubating at 94 ºC for 5 min and cDNA was synthesized using SuperScript III (Invitrogen). Second-strand cDNA was generated using dUTP-included dNTPs, instead of dTTP-included dNTPs, which was removed by Uracil DNA Glycosylase (Enzymatics) reaction after adapter ligation. During a final PCR enrichment, each library was indexed using barcode sequences-containing primers (6-mer DNA sequence) for multiplex sequencing.