GSM5453776: OA2; Glycine max; ncRNA-Seq
A high concentration of total RNA was extracted from three leaves in three biological replicates from the pBPMV-OA and pBPMV-EV- containing plants. Leaves for RNA extractions were collected from the variety Traff, prior to inoculating soybeans symptomatic for BPMV with S. sclerotiorum in one experimental replicate of the expression experiment. The third trifoliate leaves were immediately frozen in liquid nitrogen. Two grams of leaf tissue per biological replicate was ground into fine powder using liquid nitrogen in a mortar and pestle and used in RNA extractions. A phenol chloroform extraction with a LiCl purification was performed as described in Aragão et al. (2013) to yield high RNA concentrations of 1.2-2.8 ug/ul. A total amount of 2 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, 3' SR Adaptor was directly and specifically ligated to 3' end of miRNA, siRNA and piRNA. After the 3' ligation reaction, the SR RT Primer hybridized to the excess of 3' SR Adaptor (that remained free after the 3' ligation reaction) and transformed the single-stranded DNA adaptor into a double-stranded DNA molecule. This step is important to prevent adaptor-dimer formation, besides, dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5´ SR Adaptor in the subsequent ligation step. 5´ends adapter was ligated to 5´ends of miRNAs, siRNA and piRNA. Then first strand cDNA was synthesized using M-MuLV Reverse Transcriptase (RNase H–). PCR amplification was performed using LongAmp Taq 2X Master Mix, SR Primer for Illumina and index (X) primer. PCR products were purified on an 8% polyacrylamide gel (100V, 80 min). DNA fragments corresponding to 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 μL elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.