title: 16S rRNA 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. Following the Earth Microbiome Project standardized protocols, we used the 515f and 806r primer pair to target the V4 region of the 16S SSU gene. All multiplexed 16S amplicon products were pooled and sequenced on a NextSeq 2000 Instrument (Illumina, San Diego, CA) set to read a 2x150, or paired end, cycle by the Environmental Sample Preparation and Sequencing Facility (ESPSF) at Argonne National Laboratory (Lemont, Illinois).
identifiers.namespace: SUB15200108
identifiers.id: JRD189