*4.3. Extraction of RNA, Library Preparation for Transcriptome Sequencing*

To ensure the qualified samples were obtained for transcriptome sequencing, total RNA was extracted with Trizol kit (Invitrogen, Carlsbad, CA, USA) and its purity concentration and integrity detected by the Nanodrop, Qubit 2.0, Agilent 2100 method. Then, a total amount of 3 µg qualified RNA per sample was used as input material for the RNA sample preparations. According to manufacturer's recommendations, sequencing libraries were generated using NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA). The messenger RNAs (mRNAs) were separated from the total RNA by Oligo (dT) and were cleaved into short fragments at random. The first strand cDNA was synthesized by random hexamer primer, then the buffer, dNTPs, DNA polymerase I and RNase H were used to synthesize the second strand cDNAs. Lastly, the cDNAs were purified with AMPure XP beads and after end-repair and single nucleotide A (adenine) addition, the qualified cDNA libraries were constructed by PCR enrichment. After the cDNA libraries were constructed, Qubit 2.0 was used for preliminary quantification, and then the Agilent 2100 was used to detect the insert size of the libraries. After that, the Q-PCR method was used to accurately quantify the effective concentration of the libraries (effective library concentration > 2 nM) to ensure library quality. After passing the screening, high-throughput sequencing was performed with Illumina Hiseq Xten. The raw sequencing data have been submitted to the National Center for Biotechnology Information Search database (NCBI) Sequence Read Archive database with accession number SRP132610.
