4.5.1. RNA Extraction, cDNA Library Construction and Sequencing

The total RNA was extracted from NM and M using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions, and treated with an RNase-free DNase I digestion kit (Aidlab, Beijing, China) in order to remove contaminated genomic DNA. RNA degradation was measured using 1% agarose gel, RNA concentration was measured using NanoDrop 2000

spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA).

mRNA was enriched with oligo (dT)-attached magnetic beads and then the fragmentation buffer was exploited to randomly fragment the mRNA into short fragments. Using these cleaved RNA fragments as a template, the first cDNA strand was synthesized by random hexamers, then the second cDNA strand was synthesized by adding buffer, dNTPs, RNaseH and DNA polymerase I and purification of the double-stranded cDNA was done by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA). The purified double-stranded cDNA was repaired by end-to-end, added poly A tail and connected to the sequencing connector. cDNA of about 200 bp in length was selected by AMPure XP beads, and PCR amplification was performed to enrich the purified cDNA template. Finally, the libraries were sequenced using an Illumina Hiseq 2000. Sequence data were deposited in the NCBI SRA database (accession number: PRJNA579778).
