Amplicon sequencing of fungi and prokaryotes on Arabidopsis thaliana litter decomposed in Alaska
DNA was extracted from 15 mg sub-samples of an individual litterbag using the Powersoil DNA extraction kit (MoBio, Carlsbad, CA, USA). Three DNA extractions from each litterbag sample were pooled prior to PCR to obtain a better representation of the microbial community. For fungi, primers were modified versions of the primer set ITS1F and ITS2, which targets the ITS1 region of rDNA. For bacteria, primers were modified versions of the 805r and 515f primers amplifying the 16S region of rDNA. Amplicons were cleaned with the Agencourt Ampure XP kit (Beckman Coulter, Brea CA) and quantified using Qubit hs-DS-DNA kit (Invitrogen, Carlsbad CA) on a Tecan Infinite F200 Pro plate reader, reading at 485 nm excitation and 530 nm emission. PCR products for a gene region (16S or ITS) were pooled at equimolar concentrations and subject to 250 bp paired-end sequencing at the Stanford Functional Genomics Facility on an Illumina MiSeq. Both ITS and 16S amplicons for all samples were multiplexed on a single Miseq run.