B90
MiSeq of Daphnia bacterioplankton: BPK_Genotype 9_ Diet Scenedesmus_2
DNA was extracted using a PowerSoil DNA isolation kit (MO BIO laboratories), and dissolved in 20 L MilliQ water. The total DNA yield was determined using a Qubit dsDNA HS assay (Invitrogen) on 3 L of sample. 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 primers 27F and 1492R on 10 ng of template (94C - 30s; 50C - 45s; 68C - 90s; 30 cycles) using a high-fidelity Pfx polymerase (Life technologies). PCR products were subsequently purified using the QIAquick PCR purification kit (Qiagen). To obtain dual-index amplicons of the V4 region, a second amplification was performed on 5 L of PCR product using primers 515F and a slightly modified version of primer 806R for 30 cycles (94C - 30s; 55C - 30s; 68C - 60s). Both primers contained an Illumina adapter and an 8-nt barcode at the 5-end. For each sample, PCRs were performed in triplicate, pooled and gel purified using the QIAquick gel extraction kit (Qiagen). An equimolar library was prepared by normalizing amplicon concentrations with a SequalPrep Normalization Plate (Applied Biosystems) 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.
B90
SUB4684841
B90