Identification of viruses associated with alkali bees (Nomia sp.)
Individual insects were homogenized using a 1.5ml tube and pestles in 800ul of SM Buffer (0.1M NaCl, 10mM MgSO4, 50 mM Tris-HCl pH 7.4]). These were then spun down at 8,000 rpm for 5 minutes and 200ul of the supernatant were used for DNA extraction using the High Pure Viral Nucleic Acid kit (Roche Diagnostic, USA). Equal aliquots of the viral DNA from individual insects per location was pooled. The pooled DNA was then used as a template for rolling circle amplification (RCA) using the TempliPhi 2000 kit (ThermoFisher Scientific, USA). RCA products were then combined with the viral DNA at a 1:1 ratio and used to generate Illumina sequencing libraries using the Illumina DNA library prep kit (Illumina, USA). These libraries were sequenced on an Illumina NovaSeq X plus (Illumina, USA) sequencing platform.
SUB14781197
SR_NSB