16S rRNA gene amplicon sequencing of activated sludge microbiome: exposed to 100 ppb triclosan concentration
In brief, the V3-V4 regions of the 16S rRNA were amplified using primer set 341F (5- CCTAYGGGRBGCASCAG-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3)1. PCR amplifications were performed in NEBNext UltraTM II Q5 Mastermix (New England Biolabs) under standard PCR conditions. The resulting PCR amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter), and followed by indexing with unique 8 bp barcodes using the Illumina Nextera XT 384 sample Index Kit A-D (Illumina FC-131-1002). Indexed amplicons were pooled together in equimolar concentrations and sequenced on a MiSeq Sequencing System (Illumina) in paired-end mode with V3 300 bp chemistry according to the manufacturers instructions.