Platinum superfi 2 pcr master mix

The PCRs were performed using the Q5® Hot Start High-Fidelity 2× Master Mix (New England Biolabs, Inc.) in 10-µl reaction volumes containing 2 µl of DNA isolated from tick/flea using primers 5′-AAAGATGACCAAACTTGATCATTTAGAGG-3 and 5′-TCGATGAAGAACGCAGCCAGCT-3′ at a final concentration of 500 nM each which amplifies the internal transcribed spacer 1 (ITS1) region of the ticks and fleas. Thermal cycling entailed a polymerase activation step at 98 °C for 2 min; then 30 cycles of 98 °C for 10 s, 65 °C for 20 s and 72 °C for 75 s; with a final extension step at 72 °C for 5 min. The PCR products were sequenced and analyzed using the Basic Local Alignment Search Tool (BLAST) for identification.
For ticks which could not be identified using this sequenced region, primers which amplified the 16S ribosomal mitochondrial DNA (mtDNA) of the ticks were used [3 (link)]. The reactions were performed using the Platinum™ SuperFi II PCR Master Mix (Thermo Fisher Scientific) in 10-µl reactions containing 2 µl of DNA isolated from ticks with the primers at 500 nM final concentration each. Thermal cycling entailed a polymerase activation step at 98 °C for 2 min; then 30 cycles 98 °C for 10 s, 60 °C for 45 s and 72 °C for 30 s; with a final extension step at 72 °C for 5 min.

The Alt-R CRISPR/Cas9 System (Integrated DNA Technologies) and Neon Transfection System were used according to manufacturer protocols for CRISPR/Cas9 knockout in ZMEL-LD cells. Knockout was validated by harvesting genomic DNA with the DNeasy Blood & Tissue Kit (Qiagen, 69506), dgat1a and dgat1b genomic loci were PCR amplified with Platinum SuperFi II PCR Master Mix (Thermo Fisher, 12368010) and indels detected using the Surveyor Mutation Kit (Integrated DNA Technologies, 706020). Cells were incubated overnight with 100 µM oleic acid and then analyzed for lipid droplets via flow cytometry as previously described^{41 (link)}. Briefly, cells were stained for viability with 1:1000 DAPI, data acquired using the Beckman Coulter CytoFLEX Flow Cytometer (Beckman Coulter) and analyzed using FlowJo 10.8.1 (BD Biosciences).

Reverse transcription products were amplified through nested PCR using the Platinum SuperFi II PCR Master Mix (Thermo Fisher Scientific). The 25 μL reaction mixture for the first amplification reaction contained 12.5 µL of 2× Platinum SuperFi II PCR Master Mix, 0.25 µM of sense and antisense primers, and 2.5 µL of cDNA. First-round PCR products (1.5 µL) were immediately transferred to a second 25 μL-reaction mixture with Platinum SuperFi II Green PCR Master Mix. Depending on the PCR target, the primers and cycling conditions differed (Table S4). As for the cycling conditions, the manufacturer’s instructions were followed but dependent on the amplicon size the extension time was adapted: 20 seconds for the INT PCR, 30 seconds for the LTR, tRNA, and U3-U5 PCR, and 5 minutes for the Gag-U5 and Gag-U3 PCR. Electrophoresis on a 2% agarose gel was used to visualize positive reactions, followed by purification of the amplicons with the QIAquick PCR Purification kit (Qiagen).

Oligodeoxyribonucleotides (single-stranded DNA primer; IDT) were resolubilized in nuclease-free water to 100 µM. PCR amplification from plasmid or genomic DNA was performed with Platinum SuperFi II PCR Master Mix (Thermo Fisher Scientific), Q5 Hot Start High-Fidelity 2X Master Mix or 5× High-Fidelity DNA Polymerase and 5× GC Enhancer (NEB) according to the manufacturer’s protocol. After PCR, samples were gel purified from gel electrophoresis using a Monarch DNA Gel Extraction Kit (NEB).
For downstream T7 Endonuclease I assays, PCR was performed using LongAmp Hot Start Taq 2X Master Mix (NEB). Primers are listed in Supplementary Table 2.

As PHASE was not able to distinguish the diplotypes *A1 *B1a vs *A3 *B2, we established a reliable PCR-based genotyping method. Forward GSTA1-F-1336BP 5′-TGGATCCCTCAGTTTTGTAAGG-3′ and reverse GSTA1-R-1336BP 5′-TAAACGCTGTCACCGTCC-3′ oligos were used to specifically amplify the promoter region of GSTA1 using Platinum SuperFi II PCR Master Mix (ThermoFisher, USA) and the following cycling conditions: initiation for 3 min at 95 °C followed by 38 cycles at 95 °C for 30 s, 64 °C for 30 s and 72 °C for 45 s. PCR products were genotyped using forward and reverse PCR primers and Sanger sequencing service (Fasteris, Switzerland). In the case of ambiguous diplotype, PCR products were TOPO TA cloned using linearized pMiniT 2.0 vector (E1202S, New England Biolabs, USA) according to manufacturer’s recommendations. Several colonies were picked for sequencing using standard SP6 and T7 oligo and E. coli NightSeq Sanger sequencing service (Microsynth, Switzerland). This method was validated on a patient showing ambiguous genotype from Ansari et al.^{27 (link)} cohort (Table 2) and eight 1000 Genomes Project samples (Supplementary Fig. 2).

Gene-specific dsRNA was generated using the MEGAScript™ RNAi kit (Thermo Fisher, #AM1626, Waltman, MA, USA) according to the manufacturer’s instructions. Briefly, PCR primers containing the T7 promoter sequence were designed for Cx. quinquefasciatus and Cx. tarsalis PIWI genes as well as zuc and ago3 (Table 1). Hsu and CT cell cDNA generated using the High-Capacity cDNA reverse transcription kit (Thermo Fisher, #4368814, Waltman, MA, USA) was used as a template to amplify target regions for dsRNA synthesis. As a non-specific control dsRNA, GFP dsRNA was generated using a GFP containing plasmid and T7 primers (Table 1). Target regions were amplified by PCR using Platinum™ SuperFi II PCR Master Mix (Thermo Fisher, #12368010 Waltman, MA, USA) and verified with gel electrophoresis. PCR products were purified and concentrated using Zymo DNA-25 clean and concentrator kit (Zymo Research, #D4033; Irvine, CA, USA). DsRNA was generated via in vitro transcription using the MEGAScript™ RNAi kit (Thermo Fisher, AM1626, Waltman, MA, USA). Concentrations and purity of dsRNA were determined by Thermo Scientific™ NanoDrop™ One spectrophotometer and gel electrophoresis.

Single-stranded primer deoxyribonucleotides (Integrated DNA Technologies) were resolubilized (100 μM) in nuclease-free water. A PCR reaction with plasmid and genomic DNA templates was performed with Platinum SuperFi II PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer’s protocol. PCR reactions were purified by DNA agarose gel electrophoresis and subsequent DNA extraction using Monarch DNA Gel Extraction Kit (New England Biolabs (NEB)).