RNA-Seq of Escherichia coli
After the extracted RNA was tested and qualified, the ribosomal RNA (rRNA) enriched mRNA in Total RNA was removed through a probe. Subsequently, the fragmentation buffer was added to randomly break the obtained mRNA into short fragments, and the libraries were established in the manner of strand specificity (Parkhomchuk D. 2009).Using fragmented mRNA as the template and random oligonucleotides as primers, the first strand of cDNA was synthesized in the M-MuLV reverse transcriptase system. Subsequently, the RNA strand was degraded with RNaseH and in the DNA polymerase I system, Using dNTPs (replacing dTTP in dNTP with dUTP) as the raw material to synthesize the second strand of cDNA (making the second strand contain A/U/C/G), the purified cDNA was subjected to terminal repair, an A tail was added, and sequencing connectors were connected. Then, cDNA of approximately 370-420bp was screened using AMPure XP beads. Subsequently, the second strand of cDNA containing U was degraded using USER enzyme first, PCR amplification was performed, and the PCR products were purified again using AMPure XP beads. Finally, the library was obtained