title: GSM3302759: 0h_Schneiders_BioRep2; Plasmodium vivax; RNA-Seq
library_construction_protocol: For all experiments, salivary glands were dissected 14-16 days post-infected blood feed from An. dirus mosquitoes. Mosquitoes were immobilized by cold incubation (-20 C, 5 min), transferred to ice (4 C), and individually washed in a 3-step wash: 70% ethanol, 0.5 mg ml-1 penicillin- streptomycin and 1 mg ml-1 of neomycin (PSN), and 2.5 μg ml-1 FungizoneTM diluted in 1 x Dulbecco's Phosphate Buffered Solution (DPBS). Intact, clean salivary glands were collected into 100 μl of collection media with dissection time under an hour. Salivary glands were centrifuged (1600 G, 3 min) with gland disruption using plastic pestle and manually pipetting then counted using a hemocytometer. A total of 200,000 - 2,000,000 sporozoites were collected per condition. 0.5 μg - 1.0 μg total RNA of each sample was used for library preparation. Libraries were prepared by using TruSeq Stranded mRNA kit (Illumina). Library quantification was conducted by qPCR and TapeStation (Agilent Technologies) measurements. Libraries were subsequently pooled and sequenced using 300 cycle V2 MiSeq reagent kit (Illumina)