Amicon ultra centrifugal filter columns

Total RNA (large and small RNAs) was isolated from all samples by miRVana microRNA isolation Kit (AM1560, ThermoFisher Scientific). The RNA isolation protocol was followed as per the manufacturer’s instructions. A clean-up step (Amicon^® Ultra centrifugal filter columns C82301, Merck) was added after RNA elution step in order to dilute/exclude any contaminants that might interfere in NanoString profiling. 200 ng of RNA was used for NanoString miRNA profiling (Human V3 miRNA Panel).

The gene encoding the Wolbachia repressor protein was PCR amplified using primers wrpDiF/wrpDiR (Table 2), directly cloned in the pEXP-5-CT/TOPO TA expression vector and transformed into E. coli TOP10 cells (Invitrogen) according to the manufacturer’s recommendations. Following sequence verification, purified plasmid DNA was used for in vitro translation (PURExpress in vitro protein synthesis kit; NEB), and the resulting protein was concentrated using Amicon Ultra Centrifugal filter columns (molecular weight of 3,000 [3K] and 30K) (EMD Millipore, Millipore Corp., Billerica, MA). Total protein was quantified by Bradford protein assay using bovine serum albumin (BSA) as the standard (Bio-Rad).