title: Microbiota composition DCV rep 3
design: Female flies from uninfected or persistently infected stocks were collected at 12 dpe. External microbes were removed by surface sterilization using subsequent washes in 10% bleach for 5 min, 70 % ethanol for 5 min and three rinsing steps in sterile water. Since the fly microbiota is fairly stable in populations reared together, microbiota composition was analyzed in three pools of 20 flies per condition. Flies were placed in a lysis buffer (10 mM Tris-HCl (pH 8), 26 mM EDTA, 0.5% SDS, 5 mg/mL lysozyme) and homogenized with sterile pestles. Blank samples consisting of lysis buffer were processed alongside the flies and served as extraction negative controls. After incubating the lysates for 1 h at 37 C, total DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturers instruction. For each sample, 5 L of undiluted genomic DNA was used as template in a 20 L final reaction volume for amplification of the 16S rRNA gene. The primers containing locus specific sequence 8/27_Forward and 1492_Reverse were employed. PCR amplification was carried out for a total of 35 cycles. The resulting amplicons were used for library preparation with the Ligation Sequencing Kit V14 (SQK-LSK114, Oxford Nanopore Technologies) following the manufacturers instructions. Subsequently the ONT DNA libraries were sequenced on a PromethION 2 Integrated apparatus using a FLO-PRO114M flowcell.