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.