RNA-Seq of Soybean: sprout
The extraction of RNA and transcriptome determination of the test samples were completed by Beijing Beiruihekang Biotechnology Co., Ltd. Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the instructions provided by the manufacturer. The RNA concentration and purity were measured using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total of 1 g of RNA per sample was used as input material for the RNA sample preparations. The sequencing libraries were generated using the Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology Co., Ltd., Shanghai) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The first strand of cDNA was synthesized, and the second strand of cDNA was subsequently synthesized. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of the DNA fragments, NEBNext adaptors with hairpin loop structures were ligated to prepare for hybridization. The library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 l of USER Enzyme (NEB, USA) was incubated with size-selected, adapter-ligated cDNA at 37 C for 15 min, followed by 5 min at 95 C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer. Finally, the PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system. The libraries were sequenced on an Illumina NovaSeq platform to generate 150 bp paired-end reads according to the manufacturer's instructions.