StN16091DNA
DNA was extracted from 1/3 of the filter by freeze thawing (one cycle of 80 C/50 C) and bead beating using Zirconia beads (Biospec Products) in the Tissue Lyzer (Qiagen) at 50/s for 1 min. Bead beating was performed in the presence of an extraction buffer containing 10% sodium dodecyl sulfate, 500 nM Tris-HCL, and 100 mM NaCl. After bead beating, the samples were incubated at 50 C at 600 rpm for 30 min and centrifuged for 5 min at 19000  g and 10 C. The aqueous supernatant containing DNA was purified with equal volumes of phenol-chloroform-isoamyl alcohol (25:24:1vol:vol:vol) and chloroform-isoamyl alcohol (24:1 vol:vol). DNA was precipitated overnight at 4 C with two volumes of 30% polyethylene glycol in the presence of glycogen (Thermo Fisher Scientific) and centrifuged at 19 000  g at 10 C for 60 min. Nucleic acid pellets were washed once with 70% ethanol and dissolved in 50 L of TE buffer (Qiagen). 16s rRNA gene amplicon libraries of V3+V4 made with Illuminas 16S Metagenomic Sequencing Library Preparation guide, using primers Bac 341F and Bac 805R