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Total RNA served as the starting material for RNA sample preparations. Utilizing the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA, Catalog #: E7530L) in accordance with the manufacturer's guidelines, sequencing libraries were constructed, and index codes were incorporated to uniquely identify sequences for sample.In summary, mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer(5X). The first strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Subsequently, the second strand cDNA synthesis was carried out using DNA Polymerase I and RNase H. Any remaining overhangs were transformed into blunt ends through exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEB Next Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beverly, USA). Then 3 µL USER Enzyme (NEB, USA) was used with size selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Finally, PCR products were purified using the AMPure XP system, and library quality was evaluated on the Agilent 5400 system (Agilent, USA) before quantification by QPCR (1.5 nM). |
RNA-Seq |
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PCR |
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