title: Lumbricus terrestris intestinal microbiome, soil habitat was treated with mix of pesticides (10x application rate), 7 days, replicate 1, bacterial/archaeal 16S
design: The extraction of total DNA from a 0.5 mg sample of the intestines of earthworms was carried out using the FastDNA SPIN Kit for Soil (MP Biomedicals, USA) according to the manufacturer's protocol. The isolated DNA extracts were stored at -20 C. The samples of isolated DNA were diluted 500-fold. Amplification of the V4 variable region of the 16S rRNA gene was carried out in 1 round using forward and reverse primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') with two-index multiplexing of the samples (Fadrosh et al., 2014). These primers are specific both to bacteria and to archaea. The PCR products were purified using the Cleanup Mini kit (Evrogen, Russia) for DNA isolation from reaction mixtures. The concentration of the obtained 16S rRNA libraries in solution was measured on a Qubit fluorometer (Invitrogen, USA) using the Quant-iT dsDNA High-Sensitivity Assay Kit. The purified amplicons were mixed equimolarly in accordance with the concentration measurements. The quality of the resulting library prepared for sequencing was assessed by agarose gel electrophoresis. Further sample preparation and sequencing of the pooled sample was carried out using the MiSeq Reagent Kit v2 (500 cycles) and an MiSeq sequencer (Illumina, USA).