User enzyme

Total RNA was extracted from leaves of five plants that had been subjected to 10-day-drought or control treatments using Promega Total RNA Isolation System (Z3100). Then, mRNA was fragmented in the fragmentation buffer (Ambion). The first strand of cDNA was reversed transcribed with SuperScript II (Invitrogen) using random primer (TAKARA). Next, dTTP was replaced by dUTP for the synthesis of the second strand. The repaired double-stranded cDNA was ligated with Illumina TruSeq adaptor and digested using USER enzyme (NEB). Finally, 350–450 bp fragments were recovered and purified, and 18 cDNA libraries (3 accessions × 2 treatments × 3 replicates) were sequenced on the Illumina HiSeq 2500 platform.

The maximal amount of input material during PRIA is the adaptor ligation step. Based on the manufacturer's instruction, 100 ng of the DNA fragments were repaired by reacting them with the End Prep Enzyme Mix and End Repair Reaction Buffer at 20°C for 30 min and then at 65°C for 30 min. Subsequently, they were reacted with the Blunt/TA Ligase Master Mix, the NEBNext Adaptor for Illumina and Ligation Enhancer at 20°C for 15 min before finally being treated with the USER™ enzyme at 37°C for 15 min. The adaptor-containing fragments were labeled with PHPA to obtain chemically labeled fragments, after which they were denatured at 95°C for 10 min and immediately transferred to ice to acquire single stranded DNA.