Precellys 24 dual

Total RNAs were extracted from each pool using the Nucleospin RNA II extraction kit (Macherey-Nagel, Hoerdt, France). Pools were ground in 350 µL Lysis Buffer and 3.5 µL β-mercaptoethanol using the homogenizer Precellys^®24 Dual (Bertin, France) at 5500 rpm for 20 s. Total RNA per pool was eluted in 50 µL of RNase free water and stored at −80 °C until use.
When pools of abdomens were positive for virus, the RBP (head/thorax) of individual mosquitoes composing each pool were homogenized in 300 µL of DMEM with 10% fetal calf serum using the homogenizer Precellys^®24 Dual (Bertin, France) at 5500 rpm for 20 s. Then, total RNAs were extracted from 100 µL of homogenates using the Nucleospin RNA II extract kit (Macherey-Nagel, Germany) and 200 µL were conserved at −80 °C for attempts to isolate the virus. Total RNA per sample was eluted in 50 µL of RNase free water and stored at −80 °C until use.

The tissue was transferred from the cryovials into beads-filled tubes (Precellys^® Lysing Kit CK 14, Bertin, France). The CN of the animals of one age group were pooled in one bead tube (n = 4 animals and 8 CN per age group) without a medium. Pooling was necessary to generate enough mRNA for further analysis. Weighing of pooled CN was performed (Sartorius^® Handy M160, Goettingen, Germany). The pooled CN weighed less than 20 mg, regardless of age. Therefore, following the instructions of the RNeasy Mini Kit (Qiagen^®, Venlo, The Netherlands), 350 µL of RLT buffer (Qiagen^®, Venlo, The Netherlands) was added per tube. These were homogenized in two homogenizer steps (Precellys 24 DUAL^®, Bertin, France) at 6000 rpm for 30 s each. A total of 350 µL of ethanol 70% (Thermo Fisher Scientific^®, Waltham, MA, USA) was added to the resulting emulsion. Further steps were performed according to the instructions of the RNeasy Mini Kit (Qiagen^®, Venlo, The Netherlands).
Subsequently, the extracted RNA was quantified using a spectrophotometer (NanoDrop One/One^c, Thermo Fisher Scientific^®, Waltham, MA, USA), and its purity (A260/A280) was determined. At postnatal day 6 (p6), animals had approximately 350 ng/mL RNA. p12 and p24 animals had about 750 ng/mL. Only RNA with an A260/A280 ratio of 2.0 ± 0.1 was used to synthesize complementary DNA (cDNA).

In total, 998 nymphs have been collected over the 3 years [26 (link)]. As detailed in our previous studies [26 (link), 29 (link)], ticks were first washed once in ethanol 70% for 5 min and rinsed twice in sterile MilliQ water. They were then individually homogenised in 375 μL of Dulbecco’s modified Eagle’s medium with decomplemented foetal calf serum (10%) and six steel beads using the homogeniser Precellys®24 Dual (Bertin, France) at 5500 rpm for 20 s. DNA extraction was performed on 100 μL of tick homogenate, using the NucleoSpin® Tissue DNA extraction kit (Macherey-Nagel, Germany).