Transcriptomic sequencing of Escherichia coli conjugative mixed strains
Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies). Quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific) and a Bioanalyzer 2100 system (Agilent). Zymo-Seq RiboFree Total RNA Library Kit was used to remove rRNA from total RNA. Random oligonucleotides and SuperScript III were used to synthesize the first strand cDNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase HStrand-specific RNA-seqThen use RNaseH to degrade the RNA strand, and in the DNA polymerase I system, use dNTP with dUTP instead of dTTP as raw material to synthesize the second strand of cDNA. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3 ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd.