title: Amplicon sequences soil fungi
design: Subsamples (~250mg) of the bulk and rhizosphere soils were added to MoBio PowerSoil bead tubes (MO BIO, Carlsbad, CA) for DNA extraction, and frozen at -20C until all samples were processed. In order to amplify the V4 region of the bacterial 16S rRNAgene the modified 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACNVGGGTWTCTAAT-3) were used as tested in Walters et al (2015) to improve amplification of some archaeal and bacterial groups. .  PCR conditions were as follows: 98C for 30 sec, 30 cycles of 98C for 10 sec, 65C  for 10 sec, 72C for 8sec, and 72C for 5 min. The 20l PCR reactions contained 0.2l Phusion High-Fidelity DNA polymerase, 4l 5x Phusion Green HF buffer, 0.4l dNTPs (10mM), 12.4l DEPC water, 1l each of forward and reverse primer (10M), and 1l of template DNA. PCR reactions were performed in triplicate and each amplification was verified on 1.5% agarose gel.  PCR products were combined and cleaned using Axygens AxyPrep Mag PCR Clean-up Kit according to manufacturer instructions (Axygen Biosciences, Union City, CA). Concentrations of cleaned PCR products were measured using a dsDNA HS assay kit on a Qubit 3.0 Fluorometer (Invitrogen by Life Technologies, Carlsbad, CA). An equimolar mix was prepared of the cleaned bacterial and fungal products separately for submission to the University of Minnesota Genomics Center and sequenced on the Illumina MiSeq platform using V2 chemistry (2x250 PE).
identifiers.namespace: SUB5843820
identifiers.id: D31B_Bacteria