Trizol

Mosquito thoraces were combined with TRIzol (Zymo Research, Irvine, CA, USA) at a ratio of 1 mL TRIzol per 50–100 mg mosquito tissue while nematode samples were processed using a 3:1 volume ratio of TRIzol to sample. β-mercaptoethanol was added to a final concentration of 0.1%. The tissues were homogenized in a TissueLyser (Qiagen, Germantown, MD) at 50 Hz for 5 min. The homogenate was transferred to a new tube and centrifuged at 12,000 × g for 10 min at 4 °C. After incubating at room temperature for 5 min, 0.2 volumes of chloroform were added. The samples were shaken by hand for 15 s, incubated at room temperature for 3 min, then loaded into a pre-spun, phase lock gel heavy tube (5Prime, Gaithersburg, MD, USA) and centrifuged for 5 min at 12,000 × g at 4 °C. The upper phase was removed to a new tube and one volume of 100% ethanol was added prior to loading onto a PureLink RNA Mini column (Ambion, Austin, TX). The samples were then processed following manufacturer instructions, quantified using a Qubit fluorometer (Qiagen, Germantown, MD, USA) and/or a NanoDrop spectrometer (NanoDrop, Wilmington, DE, USA). The RNA was subsequently treated with the TURBO DNA-free kit (Ambion, ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

Mosquito thoraces were combined with TRIzol (Zymo Research, Irvine, CA) at a ratio of 1 ml TRIzol per 50 to 100 mg mosquito tissue, while nematode samples were processed using a 3:1 volume ratio of TRIzol to sample. For both preparations, 1 μl β-mercaptoethanol was added for every 100 μl of sample. The tissues were homogenized using a bead beater and a TissueLyser (Qiagen, Germantown, MD) at 50 Hz for 5 min. The homogenate was then transferred to a new tube and centrifuged at 12,000 × g for 10 min at 4°C. After incubation at room temperature for 5 min, 0.2 ml chloroform was added for every 1 ml TRIzol. The samples were shaken by hand for 15 s, incubated at room temperature for 3 min, loaded into a prespun Phase Lock Gel heavy tube (5Prime, Gaithersburg, MD), and centrifuged at 12,000 × g for 5 min at 4°C. The upper phase was extracted, and 1 volume of 100% ethanol was added and then loaded onto a PureLink RNA Mini column (Ambion, Austin, TX). The samples were processed according to the manufacturer’s instructions, quantified using a Qubit fluorometer (Qiagen, Germantown, MD) and NanoDrop spectrometer (NanoDrop, Wilmington, DE), and sent to the Institute for Genomic Sciences at the University of Maryland Baltimore for DNase treatment and library preparation.

Total RNA was isolated from embryonic or larval tissue by mechanically homogenizing the tissue at room temperature in 200–500 µl TRIzol (Ambion, 15596-018) followed by RNA isolation according to the TRIzol product instructions or using a Direct-zol RNA MiniPrep Plus kit (ZYMO Research Corp, 2072). DNA contamination was removed from the TRIzol-isolated RNA via enzymatic digestion with 10 U of Turbo DNase (Ambion, AM2239) at 37 °C for 15 min in a reaction tube for TRIzol-mediated extraction or on a ZYMO RNA MiniPrep spin column. DNase was removed from the RNA via organic extraction with phenol:CHCl3:IAA (isoamyl alcohol) (125:24:1) followed by CHCl3:IAA (24:1), then precipitated by adding 10% (v/v) 3 M pH 5.2 sodium acetate solution and 2.5 volumes of 100% ice-cold ethanol and cooled to − 20 °C for ≥ 20 min, then centrifuged at 16,000–20,000 RCF for 20 min. The RNA pellet was washed twice with 70% (v/v) EtOH, air-dried, and dissolved in 20–50 µl DNase/RNase-Free water. The RNA isolated using the ZYMO Direct-zol RNA MiniPrep columns was eluted in 50 μl of DNase/RNase-Free water. The final concentrations were measured at a 260 nm/280 nm absorbance on a Nanodrop 2000 spectrophotometer.