RNA-seq of Naive mosquitoes: circulating hemocytes - replicate2
To isolate RNA from hearts or abdomens, samples were homogenized in TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), extracted following the TRIzol protocol, and resuspended in Buffer RLT (Qiagen, Hilden, Germany) with 2% 2-Mercaptoethanol. The RNA was further purified using the RNeasy Micro Kit (Qiagen), DNase treated, and eluted in RNase-free water. To isolate RNA from circulating hemocytes, perfused hemolymph was collected in Buffer RLT (Qiagen) and 2% 2-Mercaptoethanol, and RNA was isolated using the RNeasy Micro Kit. The library for sequencing was prepared using 1 g of RNA and the NEBNext Ultra kit (New England BioLabs, Ipswich, MA), according to the protocol for low-input samples. Library quality and concentration were assessed using a DNA 1000 Series II kit on the 2100 Bioanalyzer.