RNA extraction
Extraction steps were conducted in a BSL-3 laboratory. The ticks were
pooled based on the geographical region before extraction, resulting a
final number of 13 pools (Table 2). The tick pools were homogenized in 2
mL reinforced tubes MK28-R from Precellys containing 1 ml of TRIzol
Reagent for cells.
Ticks were shaken two to three times for 30s using the Minilys (Bertin,
France). Between each shaking procedure the samples were placed on ice
for 1 minute. The homogenates were used for total RNA extraction. The
lysates were centrifuged at 12,000 g for 2 min at 4°C. RNA was purified
with a RNeasy mini kit (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions, including on-column DNAse treatment and
further analyzed using an Agilent Bioanalyzer. The RNA recovered in
nuclease-free water was stored at −80°C.