Instrument: BGISEQ-500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA was extracted with an RNAeasy Plus Mini kit (Qiagen, Germany) using glass beads to mechanically disrupting cells with a FastPrep (30 s ×4; M.P. Biomedical, USA). DNase treatment by RNase-Free DNase Set (Qiagen, Germany) aided to digest DNA in samples. rRNA was removed with Illumina Ribo-zero rRNA removal kit(bacteria) after total RNA was collected. Fragmentation buffer was added for interrupting mRNA to short fragments. Taking these short fragments as templates, Random hexamer-primer were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dATPs,dGTPs,dCTPs,dUTPs, RNase H and DNA polymerase I respectively after removing dNTPs. Short fragments were purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments were connected with sequencing adapters. Then, the UNG enzyme was used to degrade the second-strand cDNA, and the product was purified by MiniElute PCR Purification Kit before PCR amplification.