Nebnext ultra directed rna library preparation kit for

The sequencing library was prepared using the NEBNext®Ultra™ directed RNA library preparation kit for Illumina ® (NEB, Ipswich, MA, USA), which enabled the addition of index codes to the sequence. After the RNA samples passed the test, the ribo-zero kit was used to remove the rRNA and enrich the mRNA. On the basis of single - stranded cDNA synthesized with mRNA as the template, the synthesis of double-stranded cDNA was completed. The cDNA fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, MA, USA), the purified double-stranded cDNA was connected to the sequencing joint and the desired fragment size was selected. Finally, PCR amplification was performed to purify the product and construct the final library. Primer Index (X), Phusion high-fidelity DNA polymerase and universal PCR primers were used for the reaction. The Agilent Biological Analyzer 2100 system was used to evaluate the quality of the product and library.

Total tissue RNA was extracted using TRIzol reagent (Invitrogen™, Carlsbad, CA, USA) according to the manufacturer's manual, and the extracted RNA was tested for quality and integrity (Agilent Technologies, CA, USA). Next, rRNA was removed using the Epicenter-Ribo-zero rRNA Removal Kit (Epicentre, USA), and rRNA-free residues were removed by ethanol precipitation. Then, sequencing libraries were generated using NEBNext Ultra Directed RNA Library Preparation Kit for Illumina (NEB, USA). The library was sequenced at Novogene (Beijing, China) using the Illumina Hiseq 4000 base platform, and 150 bp paired-end reads were generated.