2.2 RNA extraction and transcriptome sequencing
Total RNA of all test samples was isolated using the Quick-RNATM MicroPrep Kit (ZYMO Research, USA) according to the manufacturer’s protocol. The integrity of the total RNA was analyzed using 1.5 % agarose gel electrophoresis (Cui et al., 2017). The quality and concentration were analyzed on NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The cDNA was synthesized by following the instructions from RTTM All-in-One Master Mix Kit (Herogen Biotech, USA). Transcriptome sequencing was performed at Novogen Co., Ltd. Beijing, China, and the samples were sequenced on the Illumina Hiseq 2500 platform. The raw reads were curated by removing adaptor sequences and reads of low quality, then assembled into unigenes using Trinity (Huang et al., 2018; Grabherr et al., 2011).