16s of Drosophila melanogaster: whole body homonogate
To test for microbiota composition of wild D. melanogaster the V4 region of the 16S rRNA marker gene was amplified from whole body homogenates of wild flies collected between 1991 and 1993 by Duane Jeffries and James Farmer, stored in ethanol, including at least some time at -20C after placing in ethanol. In 2019, DNA was extracted from individual male and female flies by a salting out procedure (Cenis, Perez, & Fereres, 1993). Briefly, fly bodies were homogenized in enzymatic lysis buffer with 20mg/ml lysozyme (Amresco, 0663) and disrupted using glass beads. Cells were then lysed via incubation with 10X extraction buffer and proteinase K. After incubation with 3 M sodium acetate, DNA was extracted from the pellet using 100% isopropanol, rinsed in 70% ethanol and resuspended in sterile TE buffer. From these extracts, the V4 region of the 16S rRNA gene was amplified as described previously (Kozich, Westcott, Baxter, Highlander, & Schloss, 2013). Amplicons were normalized using the SequalPrep Normalization kit (Invitrogen) and sequenced via 2 x 250 Illumina v2 chemistry on a HiSeq 2500 at the BYU DNA sequencing center.