Oligotex mrna mini kit

All centrifuge tubes and pipette-tips were RNase-free or were treated with DEPC. All buffers were RNase free or prepared using DEPC-treated ddH₂O. Total RNA of each sample was extracted by Trizol (Life Technology, USA) according to the manufacturer’s instructions. For the same grouping of treatments, equal amounts of the RNA from each time point were mixed together. The RNA degradome library was constructed as previously described [5 (link),37 (link)]. In brief, poly (A) RNA was isolated from approximately 200 μg of total RNA using the Oligotex mRNA mini kit (Qiagen, USA). A 5’-RNA adapter containing a Mme I recognition site in its 3’ terminus was ligated to the poly (A) RNA possessing a free 5’-monophosphate by T4 RNA ligase (Takara, China). The ligation products were purified using the Oligotex mRNA mini kit and reverse transcribed using the Oligo (dT)₁₈ primer and Superscript II reverse transcriptase (Invitrogen, USA). The first-strand cDNA was amplified for five cycles using Ex Taq DNA Polymerase (Takara, China) and the PCR products were digested with Mme I. Next, the digested products were ligated to a 3’-double-stranded DNA adaptor using T4 DNA ligase (Takara, China), and amplified by PCR for 20 cycles. The final PCR products were gel purified and subjected to SE51 sequencing using Illumina HiSeq2000 (Illumina Inc., San Diego, USA).

The 5′-RACE and 3′-RACE were carried out as described previously [31] (link). Briefly, total RNA extracted from the root of S. miltiorrhiza was purified using the oligotex mRNA mini kit (Invitrogen). 5′ and 3′ RACE was performed on mRNA using the GeneRacer kit (Invitrogen). Gene specific nesting and nested primers were designed and synthesized (Tables S1 and S2). PCR products were purified, cloned and sequenced. Based on the obtained 5′ and 3′ cDNA sequence, gene-specific forward and reverse primers were designed and synthesized (Table S3). Full-length SmRDR cDNAs were PCR-amplified, cloned and sequenced as described [31] (link).