Stem trichome small RNA-Seq from the 20 accessions

Total and Small RNA Isolation: Total RNA from stem trichomes (n = 1) were isolated using concentrated TRIzol reagent (Life Technologies). Total RNA was isolated using the E.Z.N.A.® MicroElute RNA Clean Up Kit (Omega Bio-Tek). Briefly, TRIzol Reagent (Life Technologies) and chloroform was added according to the manufacturer's instructions. After centrifugation, the RNA-containing aqueous phase was collected, mixed with 1.5 volume of 100% ethanol and applied to a MicroElute spin column (Omega Bio-Tek). The column was washed according to the manufacturers's instructions: once with RWT buffer (Qiagen), once with RPE washing buffer (Qiagen) and finally with 80% ethanol. The RNA concentration was measured on a NanoDrop ND-2000 (Thermo Scientific) and RNA integrity was examined using the 2200 TapeStation System with Agilent RNA ScreenTapes (Agilent Technologies). Total RNA was spiked with ERCCs spike-in mix 1 (Life Technologies) as well as a synthetic spike-in set for Size Range Quality Control (SRQC) together with an External Reference for Data Normalization (ERDN; Locati et al., 2015). The total RNA was divided in a large and a small fraction. The large RNA fraction was bound to a mirVana™ spin column (mirVana™ miRNA Isolation Kit, Life Technologies) according to the manufacturer's instructions. Small RNAs (<200 nts) were purified from the flow-through by adding ethanol to a final concentration of 65% (v/v) and bound to an E.Z.N.A.® MicroElute spin column. The column was washed once with RWT buffer, once with RPE buffer and once with 80% ethanol (Qiagen). The concentration and integrity of small RNA was examined as described above. Next-Generation Sequencing: Bar-coded small RNA libraries were generated according to the manufacturer's protocols using the Ion Total RNA-Seq Kit v2 and the Ion Xpress™ RNA-Seq bar-coding kit (Life Technologies). The size distribution and yield of the bar-coded libraries were assessed using the 2200 TapeStation System with Agilent D1K ScreenTapes (Agilent Technologies). Sequencing templates were prepared on the Ion Chef™ System using the Ion PI Hi-Q Chef Kit (Life Technologies). Sequencing was performed on an Ion Proton™ System using Ion PI v3 chips (Life Technologies) according to the manufacturer's instructions. References: Locati et al. 2015. Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization. Nucleic Acids Res. (2015). 43(14):e89. doi: 10.1093/nar/gkv303.