2.8 Transcriptome analysis
The RNA samples were tested for purity, concentration, and integrity. A total RNA amount of 1 μg per sample was used as input material for RNA sample preparation to ensure that qualified samples were used for transcriptome sequencing. Sequencing libraries were generated using the NEBNext®Ultra™ RNALibrary Prep Kit for Illumina® (NEB, USA), adding manufacturer and index codes to the attribute sequences of each sample. The cDNA first strand was synthesized using hexamer primers and reverse transcriptase. Subsequent synthesis of the cDNA second strand was performed using DNA polymerase I and RNase H. Once the 3’ ends of the DNA fragments were adenylated, the NEB Next Adapter was ligated and prepared for hybridization. The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments of 240 bp in length. The fragment size was then picked with 3 μL of USER enzyme (NEB, USA), and the cDNA was ligated at 37 °C for 15 min and at 95 °C for 5 min for PCR. PCR was then performed with Phusion high-fidelity DNA polymerase, universal PCR primers, and Index (X) primers. finally, the PCR products were purified and the sequences were analyzed on an Agilent Bioanalyzer the library quality of the obtained sequences was evaluated on the Agilent Bioanalyzer 2100 system.