16S rDNA of Daphnia magna gut microbiota: community after exposure to a stressor and microbiome treatment
To characterize the gut microbial communities from collected Daphnia guts, the guts of the surviving Daphnia per replicate were dissected under a stereo-microscope with sterile dissection needles at the end of the experiment and pooled per replicate (mean= 7.236 guts/sample; sd= 1.872 guts/sample; min= 2 guts; max= 10 guts; Table S6). Samples were stored under -20C until further processing. DNA was extracted using a PowerSoil DNA isolation kit (MO BIO laboratories, Carlsbad, CA, USA). DNA was dissolved in 20 L milliQ water. Because of initially low bacterial DNA concentrations in some samples, a nested PCR was applied to increase specificity and amplicon yield. The full-length 16S rRNA gene was first amplified with EUB8F and 1492R primers on 10 ng of template using a high-fidelity SuperFi polymerase (Thermofisher, Merelbeke, Belgium) for 30 cycles: 98C  10 s; 50C  45 s; 72C  30 s. PCR products were subsequently purified using the CleanPCR kit (Qiagen, Antwerp, Belgium). To obtain dual-index amplicons of the V4 region, a second amplification was performed on 5 L (=20-50 ng) of PCR product using 515F and 806R primers for 30 cycles: 98C  10 s; 50C  45 s; 72C  30 s. Both primers contained an Illumina adapter and an 8-nucleotide (nt) barcode at the 5-end. For each sample, PCRs were performed in triplicate. Afterwards the PCR products were pooled and a small volume (5 L) was loaded on a gel to check if the PCR amplified the correct fragment. The remaining volume of the PCR products were purified using the CleanPCR kit (Qiagen, Antwerp, Belgium). An equimolar library was prepared by normalizing amplicon concentrations with a SequalPrep Normalization Plate (Applied Biosystems, Geel, Belgium) and subsequent pooling. Amplicons were sequenced using a v2 PE500 kit with custom primers on the Illumina Miseq platform (KU Leuven Genomics Core), producing 2 x 250-nt paired-end reads.