SRX039175 Brew16S_Study_FVUWOJD01 â??Granular biomass samples were stored at -80 degrees C until processing. For DNA extraction, samples were ground under liquid N2, and a ~100 mg aliquot was suspended in 500 µL solution of 200 mM Tris, 200 mM NaCl, 20 mM EDTA, pH 8. This was combined with 210 microliters 20 percent SDS, 500 µL phenol:chloroform:isoamyl alcohol 25:24:1, and a 500 microliters slurry of 0.1 mm zirconia beads (BioSpec Products, Bartlesville, Oklahoma, USA). The mixture was lysed via bead beating (BioSpec bead beater, 2 min, 22 C) followed by phenol:chloroform:isoamyl alcohol extraction and precipitation with isopropanol. Extracted DNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Bacterial 16S rRNA genes were amplified using primers that were composites of 454 Life Sciences primers B (forward) and A (reverse) with conserved bacterial primers 27F and 338R. Three independent polymerase chain reaction (PCR) amplifications were performed per sample, using HotMaster PCR mix (Eppendorf), 200 µM primer, 10-100 ng template in an Eppendorf thermocycler at 95 C (2 min) followed by 30 cycles of: 95 C (20 s), 52 C (20 s), 65 C (60 s). Replicates were combined, purified using Ampure magnetic purification beads (Agencort), confirmed by gel electrophoresis, and quantified using the Quant-iT PicoGreen dsDNA kit. The purified, barcoded amplicons were pooled at equimolar ratios to a final concentration of 20 ng DNA mL-1. The single pooled amplicon mixture was sequenced at the Cornell University Life Sciences Core Laboratory Center using a Roche 454 FLX pyrosequencer.â? barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer Brew16S_Study_FVUWOJD01 AMPLICON METAGENOMIC PCR 0 Technical Read Adapter TCAG 1 barcode Technical Read BarCode AACGCACGCTAG AACTCGTCGATG AACTGTGCGTAC AAGAGATGTCGA AAGCTGCAGTCG AATCAGTCTCGT AATCGTGACTCG ACACACTATGGC ACACGAGCCACA ACACGGTGTCTA ACACTAGATCCG ACACTGTTCATG ACAGACCACTCA ACAGAGTCGGCT ACAGCAGTGGTC ACAGCTAGCTTG ACAGTGCTTCAT ACAGTTGCGCGA ACATCACTTAGC ACATGATCGTTC ACATGTCACGTG ACATTCAGCGCA ACCACATACATC ACCAGACGATGC ACCAGCGACTAG ACCGCAGAGTCA ACCTCGATCAGA ACCTGTCTCTCT ACGACGTCTTAG ACGAGTGCTATC ACGATGCGACCA ACGCAACTGCTA ACGCGATACTGG ACGCGCAGATAC ACGCTATCTGGA ACGCTCATGGAT ACGGATCGTCAG ACGGTGAGTGTC ACGTACTCAGTG ACGTCTGTAGCA ACGTGAGAGAAT ACGTGCCGTAGA ACGTTAGCACAC ACTACAGCCTAT ACTACGTGTGGT ACTAGCTCCATA ACTATTGTCACG ACTCACGGTATG ACTCAGATACTC ACTCGATTCGAT ACTCGCACAGGA ACTCTTCTAGAG ACTGACAGCCAT ACTGATCCTAGT ACTGTACGCGTA ACTGTCGAAGCT ACTGTGACTTCA ACTTGTAGCAGC AGACCGTCAGAC AGACGTGCACTG AGACTGCGTACT AGAGAGCAAGTG AGAGCAAGAGCA AGAGTAGCTAAG AGAGTCCTGAGC AGATACACGCGC AGATCGGCTCGA AGATCTCTGCAT AGATGTTCTGCT AGCACACCTACA AGCACGAGCCTA AGCAGCACTTGT AGCAGTCGCGAT AGCATATGAGAG AGCCATACTGAC AGCGACTGTGCA AGCGAGCTATCT AGCGCTGATGTG AGCGTAGGTCGT AGCTATCCACGA AGCTCCATACAG AGCTCTCAGAGG AGCTGACTAGTC AGCTTGACAGCT AGGACGCACTGT AGGCTACACGAC AGGTGTGATCGC AGTACGCTCGAG AGTACTGCAGGC AGTAGTATCCTC AGTCACATCACT AGTCCATAGCTG AGTCTACTCTGA AGTCTCGCATAT AGTGAGAGAAGC AGTGGATGCTCT AGTGTCACGGTG AGTGTTCGATCG AGTTAGTGCGTC AGTTCAGACGCT AGTTCTACGTCA ATAATCTCGTCG ATACACGTGGCG ATACAGAGCTCC ATACGTCTTCGA ATACTATTGCGC ATACTCACTCAG ATAGCTCCATAC ATAGGCGATCTC ATATCGCTACTG ATATGCCAGTGC ATCACGTAGCGG 2 linker Technical Read Linker CA 3 rRNA_primer Technical Read Primer TGCTGCCTCCCGTAGGAGT 4 Application Read Forward 454 GS FLX SRX039176 Brew16S_Study_FVUWOJD02 â??Granular biomass samples were stored at -80 degrees C until processing. For DNA extraction, samples were ground under liquid N2, and a ~100 mg aliquot was suspended in 500 µL solution of 200 mM Tris, 200 mM NaCl, 20 mM EDTA, pH 8. This was combined with 210 microliters 20 percent SDS, 500 µL phenol:chloroform:isoamyl alcohol 25:24:1, and a 500 microliters slurry of 0.1 mm zirconia beads (BioSpec Products, Bartlesville, Oklahoma, USA). The mixture was lysed via bead beating (BioSpec bead beater, 2 min, 22 C) followed by phenol:chloroform:isoamyl alcohol extraction and precipitation with isopropanol. Extracted DNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Bacterial 16S rRNA genes were amplified using primers that were composites of 454 Life Sciences primers B (forward) and A (reverse) with conserved bacterial primers 27F and 338R. Three independent polymerase chain reaction (PCR) amplifications were performed per sample, using HotMaster PCR mix (Eppendorf), 200 µM primer, 10-100 ng template in an Eppendorf thermocycler at 95 C (2 min) followed by 30 cycles of: 95 C (20 s), 52 C (20 s), 65 C (60 s). Replicates were combined, purified using Ampure magnetic purification beads (Agencort), confirmed by gel electrophoresis, and quantified using the Quant-iT PicoGreen dsDNA kit. The purified, barcoded amplicons were pooled at equimolar ratios to a final concentration of 20 ng DNA mL-1. The single pooled amplicon mixture was sequenced at the Cornell University Life Sciences Core Laboratory Center using a Roche 454 FLX pyrosequencer.â? barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer barcode rRNA_primer Brew16S_Study_FVUWOJD02 AMPLICON METAGENOMIC PCR 0 Technical Read Adapter TCAG 1 barcode Technical Read BarCode AACGCACGCTAG AACTCGTCGATG AACTGTGCGTAC AAGAGATGTCGA AAGCTGCAGTCG AATCAGTCTCGT AATCGTGACTCG ACACACTATGGC ACACGAGCCACA ACACGGTGTCTA ACACTAGATCCG ACACTGTTCATG ACAGACCACTCA ACAGAGTCGGCT ACAGCAGTGGTC ACAGCTAGCTTG ACAGTGCTTCAT ACAGTTGCGCGA ACATCACTTAGC ACATGATCGTTC ACATGTCACGTG ACATTCAGCGCA ACCACATACATC ACCAGACGATGC ACCAGCGACTAG ACCGCAGAGTCA ACCTCGATCAGA ACCTGTCTCTCT ACGACGTCTTAG ACGAGTGCTATC ACGATGCGACCA ACGCAACTGCTA ACGCGATACTGG ACGCGCAGATAC ACGCTATCTGGA ACGCTCATGGAT ACGGATCGTCAG ACGGTGAGTGTC ACGTACTCAGTG ACGTCTGTAGCA ACGTGAGAGAAT ACGTGCCGTAGA ACGTTAGCACAC ACTACAGCCTAT ACTACGTGTGGT ACTAGCTCCATA ACTATTGTCACG ACTCACGGTATG ACTCAGATACTC ACTCGATTCGAT ACTCGCACAGGA ACTCTTCTAGAG ACTGACAGCCAT ACTGATCCTAGT ACTGTACGCGTA ACTGTCGAAGCT ACTGTGACTTCA ACTTGTAGCAGC AGACCGTCAGAC AGACGTGCACTG AGACTGCGTACT AGAGAGCAAGTG AGAGCAAGAGCA AGAGTAGCTAAG AGAGTCCTGAGC AGATACACGCGC AGATCGGCTCGA AGATCTCTGCAT AGATGTTCTGCT AGCACACCTACA AGCACGAGCCTA AGCAGCACTTGT AGCAGTCGCGAT AGCATATGAGAG AGCCATACTGAC AGCGACTGTGCA AGCGAGCTATCT AGCGCTGATGTG AGCGTAGGTCGT AGCTATCCACGA AGCTCCATACAG AGCTCTCAGAGG AGCTGACTAGTC AGCTTGACAGCT AGGACGCACTGT AGGCTACACGAC AGGTGTGATCGC AGTACGCTCGAG AGTACTGCAGGC AGTAGTATCCTC AGTCACATCACT AGTCCATAGCTG AGTCTACTCTGA AGTCTCGCATAT AGTGAGAGAAGC AGTGGATGCTCT AGTGTCACGGTG AGTGTTCGATCG AGTTAGTGCGTC AGTTCAGACGCT AGTTCTACGTCA ATAATCTCGTCG ATACACGTGGCG ATACAGAGCTCC ATACGTCTTCGA ATACTATTGCGC ATACTCACTCAG ATAGCTCCATAC ATAGGCGATCTC ATATCGCTACTG ATATGCCAGTGC ATCACGTAGCGG 2 linker Technical Read Linker CA 3 rRNA_primer Technical Read Primer TGCTGCCTCCCGTAGGAGT 4 Application Read Forward 454 GS FLX