PARE of S. cerevisiae
19 g of RNA isolated from each strain, or 10 g of RNA cleaved by recombinant Rnt1, was used for two rounds of poly(A)+ RNA enrichment. T4 RNA ligase was used to ligate 5 adapters onto the 5-monophosphate ends of 3 cleavage products. NEBNext Ultra II RNA Library Prep Kit (New England BioLabs, cat. E7770, USA) was used for cDNA library preparation: Reverse transcription was performed using a 3-adapter random primer for first-strand cDNA synthesis, and the cDNAs were amplified by PCR (3 min at 95 C; 15 sec at 98 C for 15 cycles; 15 sec at 60 C, 30 sec at 72 C; and 5 min at 72 C). AMPureXP (Beckman Coulter, ref. A63882, USA) beads were used for size selection (200-400 bp), and 50-bp single-end sequencing was performed with Illumina Hiseq 2500.