title: Sample 163-16-447-WI: Location: USA: Rhinelander, Wisconsin, Host: Culex
design: A total of 163 mosquito samples were processed for microbiome analysis. Each individual female mosquito was rinsed once in 70% ethanol for 10 minutes and 5 times in sterile Dulbeccos Phosphate-Buffered Saline (DPBS) solution (Thermo Fisher Scientific, Waltham, MA) for 10 seconds and placed in a 1.5-mL microcentrifuge tube containing 100 L sterile DPBS solution. A 3.2 mm stainless steel bead was added in the tube and the whole mosquito was macerated by Tissuelyser II (Qiagen, Valencia, CA) at 28 beats/second for 4 minutes. The entire lysate was utilized for genomic DNA isolation using QIAamp DNA mini kit (Qiagen, Valencia, CA) following manufacturers instruction. Isolated DNA was reconstituted in 100 L of AE buffer and two aliquots of 50-L each were prepared and stored at -20degC until further processing. One 50-L aliquot of the resulting DNA isolate was utilized for mosquito species identification because morphological identification was impossible due to loss of morphological features during sample collection. The other 50-L aliquot of the DNA isolate was used to build a microbiome library for Illumina HiSeq sequencing at the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign