Illumina HiSeq 4000 paired end sequencing; Bulk RNAseq of Anopheles gambiae hemocytes upon Toll pathway overactivation
Bulk RNAseq of Anopheles gambiae hemocytes upon Toll pathway overactivation
Hemocyte perfusion from Anopheles gambiae mosquitoes. After hemocyte collection, cells were placed in a glass tube and allow to attach for one hour. After that, non-adherent hemocytes (mostly oenocytoids and prohemocytes) were placed in Trizol reagent and considered the Unbound fraction. Trizol reagent was added to the glass tube that contained the attached adherent hemocytes. Total extraction was carried out accroding to manufacturer's instructions dsRNA injection into Anopheles gambiae for RNA silencing. dsCactus was injected into Anopheles gambiae to overactivate the Toll pathway. dsLacZ was injected as non-related dsRNA to serve as control. Total RNA was extracted with Trizol reagent following the manufacture's instructions, using a precipitation agent (glycogen) NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) as by manufacturer instructions, except that a proprietary Sanger UDI (Unique Dual Indexes) adapters / primer system was used. Furthermore, Kapa Hifi polymerase rather than NEB Q5 was employed.