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A total of 2 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L; New England Biolabs (NEB), Ipswich, MA, USA) following the manufacturer’s recommendations, and index codes were given to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was conducted using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5×). First-strand cDNA was synthesized using random hexamer primer and RNase H. Second-strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I, and RNase H. The library fragments were purified with QiaQuick PCR Kits (ID28104; QIAGEN, Germany) and eluted with EB buffer, after which terminal repair, A-tailing, and adapter addition were implemented. Then, the products were retrieved and amplified, and the library was completed. The libraries were sequenced on an Illumina platform, and 155 base-pair paired-end reads were generated. |
RNA-Seq |
TRANSCRIPTOMIC |
PolyA |
PAIRED
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