title: RNA and ITS-seq of Ageratina adenophora rhizosphere soils
design: Soil DNA was extracted using a PowerSoil DNA Isolation Kit (Anbiosci, Shenzhen, China) according to manufacturer?s protocol. The crude DNA was purified on 1% (wt/vol) low melting point agarose gel in TAE buffer to obtain genomic DNA. The DNA bands were excised, extracted with QIAquick Gel Extraction Kits (Qiagen Inc., Mississauga, ON, Canada), and then used as the template for PCR amplification. The V4 hypervariable region of bacterial 16S rRNA gene (Biddle et al., 2008) was amplified using 2 × KAPA HiFi HotStart ReadyMix (Shanghai Dobio, Shanghai) with the primer set: 515F (GTGCCAGCMGCCGCGGTAA) (Turner et al. 1999) and 907R  (CCGTCAATTCMTTTRAGTTT) (Lane et al. 1991) with the following PCR conditions: initial denaturation at 94 C°for 3 min followed by 5 cycles of denaturing at 94 C°for 10 s, annealing at 55 C°for 15 s and extension at 72 C°for 30 s before a final extension at 72 C°for 5 min. The 16S rRNA gene applicons obtained were attached after cleanup with AMPure XP beads to dual indices and Illumina sequencing adapters using the Nextera XT Index Kit (Illumina China, Shanghai) with the following PCR (also prepared with 2 × KAPA HiFi HotStart ReadyMix) condition: initial denaturation at 95 C° for 30 s followed by 8 cycles of denaturing at 95 C° for 30 s, annealing at 55 C° for 30 s and extension at 72 C°for 30 s before a final extension at 72 C°for 5 min. The PCR was done in triplicate for each sample and PCR products were cleaned up again with AMPure XP beads, quantified using an Agilent Technologies 2100 Bioanalyzer, normalized and pooled. Then, the 16S rRNA gene applicons obtained were diluted to 4 nM with 10 mM Tris (pH 8.5) before being denatured with NaOH and sequenced (with 5% PhiX as internal control) on a MiSeq IIlumina Platform (PE250) at Genergy Biotechnology (Shanghai, China).