RNA extraction and sequencing

Wheat caryopses under control and drought stress were collected 15 days after anthesis (DAA) and frozen immediately in liquid nitrogen. The collected samples were made up of caryopses from the middle of six different wheat ears and each two ears were from three different plastic pots. Frozen tissues were pulverized in liquid nitrogen using a mortar and pestle. Total RNAs were extracted using Trizol Reagent (Shanghai Sangon Biotech Co. Ltd., China) following the manufacturer's instructions. The purity, concentration, and integrity of RNA samples were assayed using Nanodrop, Qubit2.0, and Agilent 2,100 bioanalyzer, respectively. Two small RNA libraries were constructed using the small RNA Sample Pre Kit. Briefly, the extracted total RNA was ligated to 5′ and 3′ adapters using T4 RNA Ligase 1 and T4 RNA Ligase 2, respectively. Subsequently, the cDNA was synthesized by reverse transcription and amplified using polymerase chain reaction (PCR). Then the products were purified via PAGE to attain small RNA libraries. Finally, the quality control of libraries was performed using Qubit 2.0, Agilent 2,100 bioanalyzer, and quantitative PCR. The resulting RNA was sequenced by Nanjing Genepioneer Biotechnologies using the Illumina HiSeq2500 high-throughput sequencing platform.