title: 16S-seq of Drosophila melanogaster Oregon-R-modENCODE, Adult,  Feces, Pesticide treatments
design: Feces was collected by swabbing the walls of fly cages.  DNA was isolated from fecal swabs using the PureLink Microbiome DNA Purification Kit (Invitrogen).  PCR amplification of the V4 region of the 16S rRNA gene was performed with: 10X PCR Buffer without MgCl2 (1X); 50mM MgCl2 (1.5mM); RTPCR grade water, 10mM dNTP mix (0.3mM each); 16s RNA gene primers (V4 region) 515F (0.2M, 5-GTGCCAGC M GCCGCGGTAA-3) and 806R (0.2M, 5-GGACTAC HV GGGT W TCTAAT-3); Platinum Taq DNA polymerase (1U), fecal DNA prep (<500ng).  PCR was performed on ABI 9600 thermal cyclers using the following conditions: 94C for 3 minutes; 35 cycles of (94C for 45 seconds, 50C for 1 minute, 72C for 1.5 minutes); 72C for 10 minutes; 4C hold.  Sequencing libraries were made using the NEBNext Ultra DNA Library Prep Kit for Illumina Rev 3.0 with the following modifications: 5l PCR product plus 50.5 l nuclease free water were used for the end prep stage; cleanup of adaptor-ligated DNA proceeded without size selection; 9 cycles of PCR enrichment were performed using NEBNext Q5 Hot Start HiFi PCR Master Mix.  Libraries were sequenced on an Illumina MiSeq using the 500-cycle (2x250nt) MiSeq Reagent Kit v2 according to manufacturers instructions