Dulbecco s phosphate buffered saline solution dpbs

A total of 163 mosquito samples were processed for microbial analysis (Table 1). Each individual female mosquito was rinsed once in 70% ethanol for 10 min and 5 times in sterile Dulbecco’s phosphate-buffered saline (DPBS) solution (Thermo Fisher Scientific) for 10 s and placed in a 1.5 ml microcentrifuge tube containing 100 μl 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, USA) at 28 beats/s for 4 min. The entire lysate was utilized for genomic DNA isolation using QIAamp DNA mini kit (Qiagen) following the manufacturer’s instructions. Isolated DNA was reconstituted in 100 μl of AE buffer and two aliquots of 50 μl each were prepared and stored at -20 °C until further processing. One 50-μl aliquot of the resulting DNA isolate was utilized for mosquito species identification because morphological identification was impossible due to the loss of morphological features during sample collection. The other 50-μl 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.