16S sequencing of Berghia stephanieae: C2 (cobalt 3mg/L)
To generate a cDNA template, 50 ng of each RNA sample was used with the LunaScript RT SuperMix Kit (NewEngland Biolabs, UK)(NewEngland Biolabs, UK) following the manufacturers recommendations. From the obtained cDNA, 5 l were used as PCR template in a total volume of 20 l using 10 l Go-Taq polymerase master mix (Promega, USA) and 10 mM of forward and reverse primers specific for the V1V2 region of the bacterial 16S rRNA gene attached to Illumina adapter sequences (V1V2forward: 5-TCT TTC CCT ACA CGA CGC TCT TCC GAT CTA GAG TTT GAT CCT GGC TCA G-3; V1V2reverse: 5GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TTG CTG CCT CCC GTA GGA GT-3; adapter sequence underlined). Amplification was performed in a three-step PCR reaction on a Mastercycler gradient (Eppendorf, Germany) as follows: 15 min initial denaturation at 95 C, 30 cycles of 1 min denaturation at 94 C, 1 min primer annealing at 54 C and 1min of elongation at 70 C, followed by 10 min of final elongation at 70 C. Successful amplification was verified using gel-electrophoresis and PCR products purified using 0.7 volume of AMPure XP beads (Beckman Coulter, USA). Subsequently, 5 l of purified PCR products were used as a template in a 20 l PCR reaction including 10 l of double concentrated Q5 High-Fidelity polymerase master mix (New England Biolabs, UK), 10 mM of Illumina sequencing adapters containing TrueSeq indexes. Amplification was performed in a 3-step manner with an initial denaturation of 5 min at 95 C, 8 cycles of 1 min at 94 C denaturation, 1 min at 52 C annealing, and 1 min at 70 C for elongation, followed by 10 min of final elongation at 70 C. PCR products were purified using 0.7 volume of AMPure XP beads and quantified using a qubit (ThermoFisher, USA). All samples were concentrated equimolar to 5 nM and mixed equally to generate the cluster mix for sequencing 300 cycles on a MiSeq plattform at the University of Saabrcken-non-codinglibraries,rRNAisremovedusingtheRibo-ZerokitthatleavesthemRNA.First,themRNAisfragmentedrandomlybyaddingfragmentationbuffer,thenthecDNAissynthesizedbyusingmRNAtemplateandrandomhexamersprimer,afterwhichacustomsecond-strandsynthesisbuffer(Illumina),dNTPs,RNaseHandDNApolymeraseIareaddedtoinitiatethesecond-strandsynthesis.Second,afteraseriesofterminalrepair,Aligationandsequencingadaptorligation,thedouble-strandedcDNAlibraryiscompletedthroughsizeselectionandPCRenrichment.