4.4.1. RNA Isolation and Sequencing

RNA was isolated from WT1, WT2, WT3, RT1, RT2, and RT3 samples using an RNeasy Plant Mini kit (QianGen). Extracted RNA was checked for degradation and contamination using 1% agarose gels and a NanoPhotometer spectrophotometer (Implen, CA, USA), respectively, and then quantified with a Qubit RNA Assay kit on a Qubit 2.0 fluorometer (Life Technologies). RNA integrity was assessed on a Bioanalyzer 2100 system (Agilent) using the supplied RNA Nano 6000 assay kit.

For sequencing library construction, first and second-strand cDNA synthesis was carried out using 3 µg of RNA and a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following the manufacturer's instructions. After purification using an AMPure XP system (Beckman Coulter, Beverly, MA, USA), the synthesized strands were subjected to three-end adenylation and the addition of a poly-A tail and a NEBNext adapter. Next, 150–200-bp adapter-ligated fragments were preferentially selected using AMPure XP beads and amplified by PCR. The quality of the enriched cDNA library was assessed on an Agilent Bioanalyzer 2100 system. Clusters were generated from the qualified library using a cBot Cluster Generation System with a TruSeq PE Cluster kit v3-cBot-HS (Illumina, San Diego, CA, USA) and then sequenced on an Illumina Hiseq platform to generate 125-bp/150-bp paired-end reads (raw reads).
