Dneasy blood tissue

DNA was isolated from untreated CT26 cells using the DNeasy Blood & Tissue (Qiagen) isolation kit as per the manufacturer’s instructions. The qualified genomic DNA sample was randomly fragmented by Covaris technology, and the size of the library fragments was mainly distributed between 150 bp and 250 bp. The end repair of DNA fragments was performed, and an "A" base was added at the 3′-end of each strand. Adapters were then ligated to both ends of the end repaired/dA–tailed DNA fragments for amplification and sequencing. Size-selected DNA fragments were amplified by ligation-mediated PCR, purified, and hybridized to the BGI exome array for enrichment. Nonhybridized fragments were then washed out and captured products were circularized. The rolling circle amplification was performed to produce DNA Nanoballs. Each resulting qualified captured library was then loaded on BGISEQ-500 sequencing platforms, and we performed high-throughput sequencing for each captured library to ensure that each sample met the desired average sequencing coverage.

Genomic DNA was extracted from two first-generation adult mites per fruit, each representing an experimental sample. For this, the DNeasy Blood & Tissue (QIAGEN, Germantown, MD, USA) kit was used following the manufacturer’s instructions. A fragment of the gene COI was amplified using the primers DNF (TGA TTT TTT GGT CAC CCA GAA G) and DNR (TAC AGC TCC TAT AGA TAA AAC) [24 ]. PCR reactions were done in a 25 μL reaction volume containing 2.5 μL of buffer 10X (600 mM Tris-SO4 (pH 8.9), 180 mM ammonium sulphate), 1 mM of MgCl2, 0.2 μM of each primer, 0.2 mM of dNTP’s, 0.5 μL of Taq DNA polymerase (Quiagen, GmbH, Hilden, Germany) and 5 μL (approx. 20 ng) of DNA. PCR amplifications were done using a MyCycler (BIO-RAD Laboratories Inc., Hercules, CA, USA). Thermal conditions used were as follows: one cycle of 4 min at 94°C, followed by 35 cycles of 60 s at 94°C, 60 s at 54°C and 60 s at 72°C with a final extension at 72°C for 5 min. PCR products were visualized on 1% agarose gels in 1X TAE. GelPilot 100 bp Plus (QIAGEN, GmbH, Hilden, Germany) size markers were used. The gels were stained with ethidium bromide (0.1 μg mL^–1) and photographed. All PCR products were sent to the company Macrogen Inc. (South Korea) for direct sequencing.

Clone CL Brener (CLB) (T. cruzi Discrete Typing Unit (DTU) VI- lineage TcVI) (Zingales et al., 1997 (link); Zingales et al., 2009 (link); Zingales et al., 2012 (link)) and the G strain (T. cruzi DTU I - lineageTcI) (Yoshida, 1983 (link); Briones et al., 1999 (link); Zingales et al., 2009 (link); Zingales et al., 2012 (link)) in axenic cultures at 28°C in liver-infusion tryptose medium (LIT) containing 10% fetal calf serum. Clone D11, which also belongs to the DTU I - lineageTcI (Lima et al., 2013 (link)), was isolated from the G strain by Santori (1991) using the protocol described by Lima and Villalta (1989) (link). Log-phase epimastigotes were washed in phosphate buffered saline (PBS) and collected by centrifugation. Genomic DNA extraction was performed with the DNeasy Blood & Tissue (Qiagen) kit using 5x10⁷ cells/mL according to the manufacturer’s instructions.

Samples from bioreactor inoculated with MOI 0.1 and MOI 1.0 were submitted to qPCR analysis to compare the viral DNA quantification to OBs quantification. The OBs obtained from 3 and 7 dpi (MOI 0.1) or at 3, 7, 11 and 14 dpi (MOI 1.0) were dissolved in an alkaline solution and used to extract DNA with DNeasy Blood & Tissue (Qiagen), following the manufacturer’s instructions. The DNA was quantified with a low-DNA-mass ladder (Invitrogen) in 0.8% agarose gel electrophoresis. SfMNPV-6nd OBs obtained from larvae were extracted and serially diluted for absolute quantification. The qPCR for SfMNPV with specific primers for the sf32 gene was carried out in a Rotor gene 5plex HRM platform (Qiagen), according to [17 (link)].

Five weeks after birth, hDPP4 mice were individually selected, their genomic DNA was extracted (Supplementary Fig. 3). Genomic DNA was isolated from mouse-tail tissue using the commercial kit DNeasy^® Blood & Tissue (Qiagen) following the protocol provided by the manufacturer. The hDPP4 gene was identified by PCR using a specific primer set (forward primer: 5′CGC TAT TAC CAT GGT GAT GCG 3′, reverse primer: 5′AGC TGT AGC ATC ATC TGT GCC 3′), obtaining an amplicon size of 984 bps. The following PCR conditions were used: 94 °C for 5 min followed by 35 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and final extension at 72 °C for 10 min. The single amplified product was confirmed using agarose gel electrophoresis.