Cloacal microbiome of Passer domesticus: adult male post-captivity neophobic
We used QIAamp PowerFecal Pro DNA Kits (Qiagen #51804) to extract total microbial DNA from cloacal swabs as well as from four control samples (handled in the same manner but not inserted into a cloaca). Next, we quantified DNA concentrations using a NanoDrop 2000 (Thermo Fisher Scientific). To verify the presence of DNA in low yield (<10 ng/l) samples, we amplified the 16S region of the rRNA gene in bacterial DNA using Bakt_341F and Bakt_805R primers (CCTACGGGNGGCWGCAG and GACTACHVGGGTATCTAATCC, respectively) (Herlemann et al. 2011). We performed PCR in a C1000 Touch Thermocycler (Bio-Rad) using 10 l volumes, which included: 5 l DreamTaq Green PCR Master Mix (Thermo Fisher Scientific K1081), 2 l template DNA, 2.88 l D/RNAse free water, and 0.06 l each of Bakt_341F and Bakt_805R primers. Thermocycling conditions included an initial denaturing step of 94oC for 3 min, followed by 30 cycles of: 94oC for 30 s, 50oC for 30 s, and 72oC for 45 s, and a final extension of 72oC for 10 min. We visualized the amplicons on a 1% agarose gel stained with 2.5 l RedSafe Nucleic Acid Staining Solution (Bulldog Bio) under UV light after electrophoresis for 40 min at 100V/2A. All low-yield samples showed clear amplification of the 16S rRNA gene and were submitted for sequencing to Michigan State Universitys Research Technology Support Facility for barcoding and sequencing on a Miseq platform using a v2 reagent cartridge for a 2 x 250 bp paired-end format using primers 515f and 805r following the Kozich et al (2013) protocol.