18S rRNA gene DNA seq of microbialite microbiome: Great Salt Lake recovery experiments
DNA samples were extracted using Qiagen DNeasy PowerSoil kits (Qiagen, Germantown, Maryland, USA) according to kit directions, except that an initial extraction step was performed in the field immediately after sample collection using a hammer drill-based 3D-printed field bead beater (Smash-O-Tron), bead tubes were then flash-frozen on dry ice and stored frozen at -20C until the remaining extraction steps were completed. Aliquots of extracted DNA were sent to WrightLabs (Huntingdon, Pennsylvania, USA) for library preparation and sequencing.  DNA concentrations were quantified using an Invitrogen Qubit 4 Fluorometer and 1X Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). All 18S rRNA illumina-tag PCR reactions were performed on DNA extracts per the Earth Microbiome Projects protocol for 18S (Amaral-Zettler et al. 2009; Stoeck et al. 2010). A negative control (PCR grade nuclease-free water) was processed in parallel with the samples for PCR amplification. PCR products were pooled and gel purified on a 2% agarose gel using the QIAquick Gel Purification Kit (Qiagen, Frederick, Maryland, USA). No negative controls had amplification and were therefore not added to the sample pool. Before sequencing, the purified pool was quality checked using an Agilent 2100 BioAnalyzer and Agilent DNA High Sensitivity DNA kit (Agilent Technologies, Santa Clara, California, USA). The purified pool was stored at -20C and then it was sequenced in-house using an Illumina MiSeq v2 chemistry with paired-end 150 base pair reads.