GSM7780762
Dissection of mosquito midguts and salivary glands was performed in situ on adult females at 7 days post-blood meal. To determine infection levels, mosquito guts were dissected 7 days after blood feeding and were stained with 2% Mercurochrome before microscopic examination. Tissues were maintained in ice-cold Schneider's insect culture medium (Sigma-Aldrich) and fresh tissues were immediately processed for chromatin and RNA analyses Total RNA was extracted from fresh mosquito tissues (~25 midguts and ~ 50 salivary glands) using the mirVana™ RNA Isolation Kit (Ambion®) according to the manufacturer protocol and used for mRNA library preparation. RNA concentration was quantified using a Qubit® 2.0 Fluorometer, and RNA integrity was determined with an Agilent 2100 Bioanalyzer. Illumina libraries were prepared and sequenced at the HudsonAlpha Institute for Biotechnology, using an Illumina HiSeq2000 sequencer, standard directional RNA-seq library construction, 50 bp paired end reads with ribosomal reduction (RiboMinus™ Eukaryote Kit, Ambion®)