Illumina sequencing of targeted amplicon 16SrDNA V3-V4 region for treated wastewater sample.
Total environmental RNA from the sample of interest was isolated using Environmental DNA/RNA Extraction Kit (Eurx, Gdask, Poland) following manufacturers instructions. The quality of the isolated RNA was further improved by the use of the Clean-up RNA Concentrator kit (A&A Biotechnology, Gdask, Poland), which included a DNAse based DNA removal step. The reverse transcription was carried out with the use of  RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Sunnyvale, CA, USA) containing random hexamer primers according to the manufacturers instructions. Amplification of conserved bacterial 16SrRNA gene fragment covering V3 and V4 regions was done for cDNA samples in triplicate with use of the gene specific primers extended on 5 end with Nextera adaptors (Klindworth et al. 2012)(Illumina, Sand Diego, USA). Obtained amplicons of the size c.a. 450bp  were checked on 1% agarose gel and purified by Ampure XP magnetic beads (Beckman Coulter, Brea CA, USA). Amplicon libraries were pooled in equimolar ratio and indexed according to Nextera indexing strategy by PCR (Illumina, Sand Diego, USA). Sample indexing allowed pooling of amplicons for sequencing run and further extraction of the sample sequence reads from large batch of sequencing results data. 16S amplicons were sequenced on MiSeq sequencer in the DNA Sequencing and Oligonuleotide Synthesis Laboratory IBB PAS in paired end mode using 600 cycle v3 chemistry kit (Illumina, San Diego, USA).