RNA-Seq of ZM4: ATCC 31821 at  pH 3.6
rRNA was  removed with kit after total RNA was collected from prokaryocyte.  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. At last, the library could be  sequencing using filter.