RNA-seq of Vibrio algionlyticus: no cycle lipopeptide treatment
A total amount of 3 g RNA per sample was used as input material for the RNA samplepreparations. Sequencing libraries were generated using NEBNext Ultra DirectionalRNA Library Prep Kit for Illumina (NEB, USA) following manufacturersrecommendations and index codes were added to attribute sequences to each sample.Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.For prokaryotic samples, rRNA is removed using a specialized kit that leaves the mRNA.Fragmentation was carried out using divalent cations under elevated temperature inNEBNext First Strand Synthesis Reaction Buffer5X. First strand cDNA wassynthesized using random hexamer primer and M-MuLV ReverseTranscriptaseRNaseH-. Second strand cDNA synthesis was subsequently performedusing DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP werereplaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, NEBNext Adaptorwith hairpin loop structure were ligated to prepare for hybridization. In order to selectcDNA fragments of preferentially 150~200 bp in length, the library fragments werepurified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 l USEREnzyme (NEBUSA) was used with size-selected, adaptor-ligated cDNA at 37C for 15min followed by 5 min at 95C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, productswere purified (AMPure XP system) and library quality was assessed on the AgilentBioanalyzer 2100 system.