R50M1_1_Roots_ 50 zeb_ 1 month in soil_ rep1
A total amount of 3 g 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 manufacturers recommendations and index codes were added to attribute sequences to each sample. Briefly, NEB 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. 5ends adapter was ligated to 5ends 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 a 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 L elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.