title: ITS2 amplicon data from red alder leaves
design: DNA was extracted from 90 mg of leaf tissue from each leaf pack using the DNeasy Plant Pro Kit (Qiagen, Hilden, Germany) with the following modifications describe below. Step 1 was modified to 450 L of Solution CD1 and 50 L of Solution PS. We modified Step 2 so that all samples were homogenized using a Mini BeadBeater 24 Disrupter (BioSpec, Bartlesville, OK) for 45 seconds at 2400 rpm immediately followed by 1 minute on ice. This step was repeated once more before proceeding to the next step to make sure all leaf tissue was sufficiently lysed. Lastly, we modified Step 16 so that 50 L of Buffer EB was used, where the buffer sat on the filter membrane for 5 minutes prior to the final elution centrifugation step. DNA samples were then immediately flash frozen with liquid nitrogen and stored at -80C until downstream use. However, to estimate fungal relative abundance and richness, we used the primer set 5.8S-Fun and ITS4-Fun14 to target the 5.8S-ITS2 gene region that maximizes coverage within the Kingdom of Fungi while minimizing nonfungal sequences. Pooled amplicon products were sequenced on an Illumina MiSeq Instrument set to a 2x250 cycle at the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory.
identifiers.namespace: SUB15205482
identifiers.id: JRD782