title: 16S amplicons: Spelt sourdough - 48 h
design: For culture-independent analyses, 10 g of each sourdough sample was homogenized in a final volume of 100 ml sterile physiological salt for 5 min at 24,000 rpm. This suspension was centrifuged at 4 C for 5 min at 200g. The supernatant was collected and centrifuged at 4 C for 15 min at 5,000g. The cell pellet was frozen in liquid nitrogen and stored at -20 C until further use. The frozen pellet was thawed on ice and washed with 1 ml of TES buffer (25 mM Tris, 10 mM EDTA, 50 mM sucrose).  Prior to DNA extraction, enzymatic lysis of bacteria was performed as previously described for kefir grains (lysis C) (Kowalczyk et al. 2012; doi: 10.1017/S0022029911000677) or oscypek samples (Alegria et al. 2012; doi:10.1128/AEM.06081-11). DNA extraction was performed using the phenol/chloroform method followed by incubation with RNase A. After isolation, DNA quality was checked by electrophoresis of the sample on 0.8% agarose gels. Amplification of the conserved bacterial 16SrRNA gene fragment covering the V3 and V4 regions was performed in triplicate using the gene-specific primers 16S_V3-F 341-357F  and 16S_V4-R 785-805R (Klindworth et al. 2013; doi:10.1093/nar/gks808). Obtained amplicons of size ca. 450 bp (16S) were checked on 1% agarose gels and purified using Ampure XP magnetic beads. The amplicon libraries were pooled in equimolar ratios and indexed according to the Nextera indexing strategy by PCR. Sample indexing allowed pooling of amplicons for sequencing runs and further extraction of the sample sequence reads from large batches of sequencing data. The amplicons were sequenced on a MiSeq sequencer in paired-end mode using a 600-cycle v3 chemistry kit (Illumina).