RNAseq library preparation
The plant total RNA was extracted using the RNAprep Pure Plant Kit
(Tiangen, Beijing, China) according to the instructions provided by the
manufacturer. RNA concentration and purity was measured using NanoDrop
2000(Thermo Fisher Scientific, Wilmington, DE). RNA integrity were
assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer
2100 system (Agilent Technologies, CA, USA). A total amount of 1 μg RNA
per sample was used as input material for the preparations of RNA
samples. Sequencing libraries were generated using Hieff NGS Ultima
Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology
(Shanghai) Co., Ltd.) following 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.
First strand cDNA was synthesized, and second strand cDNA synthesis was
subsequently performed. Remaining overhangs were converted into blunt
ends via exonuclease/polymerase activities. After adenylation of 3’ ends
of DNA fragments, NEBNext Adaptors with hairpin loop structure were
ligated to prepare for hybridization. The library fragments were
purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then
3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-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. At last, PCR products were purified
(AMPure XP system) and library quality was assessed on the Agilent
Bioanalyzer 2100 system. The libraries were sequenced on an Illumina
NovaSeq 6000 platform to generate 150 bp paired-end reads, according to
the manufacturer’s instructions.