title: DNA-seq of DNA extraction kit
design: A total of 246 samples were processed for DNA extraction. The samples were split into Bacterioplankton (101 samples) and Daphnia (Juveniles: 41 samples, Adults: 102 samples). For bacterial community analysis of MPs lab exposure samples, the DNA was extracted with Powersoil Pro (Qiagen) and NucleoSpin (Macherey-Nagel) DNA isolation kit for bacterioplankton and Daphnia samples, respectively (Supplementary table). In both cases, the manufacturer's protocol was followed. The extracted DNA was then resuspended in 20 L of the respective DNA kit's elution buffer, and the overall DNA concentration was determined using a NanoPhotometer N60. Initially, the full-length 16S rRNA gene was amplified using forward EUB8F and reverse 1492R primers (Table 1) with 10 ng of template DNA and high-fidelity SuperFi polymerase (Life Technologies) across 30 cycles of denaturation at 98C for 10 seconds, annealing at 50C for 45 seconds, and extension at 72C for 30 seconds. The PCR products were purified using the CleanPCR kit (CleanNA), according to the manufacturer's instructions, and DNA concentrations were determined with a Nanodrop spectrophotometer. A second amplification was then conducted on 10 ng of the purified PCR product using forward 515F and reverse 806R primer pair (Table 1) primer pair for 30 cycles, under the same thermal cycling conditions as before (Kozich et al., 2013)1. The second step PCR primers incorporated both an Illumina adapter sequence and an 8-nucleotide barcode at the 5' end for multiplex sequencing. The last PCR amplifications reactions were conducted in triplicate for each sample, pooled, and the correct size of the amplicon verified on a 1.5% agarose gel. The amplicons were purified using the CleanPCR kit (CleanNA) and sequenced on the Illumina MiSeq platform using a v2 PE500 kit producing 250-nt paired-end reads. To prepare an equimolar library for sequencing, amplicon concentrations were normalised using a SequalPrep Normalisation Plate (Applied Biosystems) and pooled accordingly.