Degradome of cucumis sativus
Libraries for 5 parallel analysis of RNA end (PARE) were constructed using poly (A+) RNA (1000 ng) purified with GenElute Direct mRNA Miniprep Kit (Sigma-Aldrich). A 5 RNA oligonucleotide adaptor (PARE 5 Adapter) containing a Mme I recognition site was ligated to the 5-phosphate of the truncated poly (A+) RNA by T4 RNA ligase. The ligated products were purified by ethanol precipitation and subjected to a reverse transcription reaction with an oligo dT primer with a known tail (dT primer). Following RNA degradation by alkaline lysis, the cDNA was amplified by PCR using adaptor and oligo dT-specific primers (I5(1)-F and GR3, respectively), digested with Mme I and ligated to a 3 double DNA adaptor (dsDNA-top and dsDNA-bottom). The ligated products were separated by electrophoresis in polyacrylamide, and those with the expected size were gel-purified and amplified with I5(2)-F and the corresponding indexed I7-R. PCR products of the expected size were gel-purified and subjected to sequencing by Illumina technology.