RNA-Seq of epiphytic microbes associated with leaves of Ranunculus acris and Trifolium pratense
Epiphytic bacterial DNA was extracted using ZymoBIOMICS kit with an initial vortexing step for 10 min. Amplicon PCRs were performed with a dual-indexing strategy according to Kozich et al. (2013). We used USEARCH v11.0.667 for the complete sequence analysis (Edgar, 2010). FASTQ sequences data were merged and length truncated with a minimum read length of 250 bp and maximum sequence differences of 10 base pairs of each sequence. After quality filtering, dereplication, singleton exclusion and chimera removal, sequences were denoised and dereplicated into amplicon sequence variants (ASVs) using the Unoise3 algorithm (Edgar, 2016). The following taxonomy assignment was executed based on the RDP v16 reference database using bootstrap levels of 0.8 (Edgar, 2016).