title: Metabarcoding of prokaryotic sediment communities
design: For prokaryotic community analysis, 2 l RNA extract of each replicate were reverse transcribed into cDNA using the random primers supplied by the iScript synthesis kit (BioRad). The hypervariable V4 region of the 16S rRNA gene was used as target marker gene employing the primer pair 515Fm (5-GTGYCAGCMGCCGCGGTAA-3) and 806Rm (5-GGACTACNVGGGTWTCTAAT-3, Walters et al., 2016). For the amplification of the marker gene, 2 l of cDNA were used as a template for each replicate. The PCR protocol consisted of an initial activation step at 98 C for 30 s, followed by 26 cycles consisting of 98 C for 10 s, 63 C for 30 s and 72 C for 30 s, and a final extension for 5 min at 72 C. To minimize PCR bias, three individual PCR reactions for each of the samples were prepared. Afterwards, these replicate samples were pooled and subjected to library preparation using the Next Ultra DNA Library Prep Kit for Illumina (NEB, United States). The quality of the final libraries was assessed with an Agilent Bioanalyzer 2100 system. Libraries were sequenced on an Illumina MiSeq platform, generating 2x300 nt paired-end reads