title: 16S rDNA of Daphnia magna microbiota: community after exposure to stressor
design: At the end of the experiment, surviving individuals were dissected and D. magna guts and bodies were collected separately to determine the microbial community composition of each genotype and body part via amplicon sequencing. In addition, bacterioplankton samples of the medium were taken by filtering 200 mL of the medium over a 0.22 m filter. Samples were collected on ice (to prevent microbial community shifts and to preserve DNA) in an Eppendorf tube containing 10 L sterile Milli-Q. For each exposure, individuals were pooled per genotype and maternal line. Amplicon sequencing was performed according to Houwenhuyse et al., 2021. DNA was extracted by the Qiagen PowerSoil DNA isolation kit and dissolved in 20 L MilliQ water. To determine the total DNA yield, 1 L of sample was used in an Invitrogen Qubit dsDNA HS assay. Increased specificity and amplicon yield were obtained by using nested PCR. The entire 16S rRNA gene was amplified for 30 cycles (98 C for 10 s; 50 C for 45 s; 72 C for 30 s) with the Life Technologies SuperFi high fidelity polymerase and the EUB8F and 1492R primers on 10 ng of template. The PCR product was purified with the QIAquick PCR purification kit. In a second amplification round, 5 L (20-50 ng) of the PCR product was amplified for 30 cycles (98 C for 10 s; 50 C for 5 s; 72 C for 30 s) with the 515F and 806R primers to obtain amplicons of the V4-region with a dual index. The latter two primers contained an 8-nucleotide barcode, as well as an Illumina adapter at their 5-end. PCRs were performed in triplicate and were pooled and gel-purified for each sample with the QIAquick gel extraction kit. To prepare an equimolar library, an Applied Biosystems SequalPrep Normalization Plate was used to normalize amplicon concentrations, after which the library was pooled. Amplicon sequencing was performed using a v2 PE500 kit with custom primers on the Illumina Miseq platform, which resulted in two 250-nucleotide paired-end reads for each of the 36 samples.