16S rDNA of Daphnia magna microbiota: community after exposure to stressor
The five D. magna taken from each treatment at each timepoint 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, these five individuals were pooled per genotype and maternal line. Amplicon sequencing was performed according to (41). We extracted DNA using the DNA insect kit (Macherey Nagel) following the manufacturers protocol and eluted in 20 L MilliQ water. To determine the total DNA yield, 0.5 L of sample was used on Nanodrop. Increased specificity and amplicon yield were obtained by using nested PCR (i.e. first amplifying the entire 16S rRNA gene followed by amplifying the V4 region from this PCR product). 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 (5  AGA GTT TGA TCM TGG CTC AG  3) and 1492R (5  GGT TAC CTT CTT ACC ACT T  3) primers on 25 ng of template. The PCR product was purified with the Clean PCR kit from GC Biotech. In a second amplification round, 10 ng of the PCR product was amplified for 30 cycles (98 C for 10 s; 55 C for 45 s; 72 C for 30 s) with the 515F and 806R primers to obtain amplicons of the V4-region with a dual index, as the latter two primers contained an 8-nucleotide barcode, as well as an Illumina adapter at their 5-end. Internal PCRs were performed in triplicate (i.e. technical replicates) and 5 l was used to check if the fragments were correct on gel, the rest of the sample has been purified with Clean PCR kit (GC Biotech). To prepare an equimolar library, an Applied Biosystems SequalPrep Normalization Plate (Invitrogen) 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 82 samples.