title: GSM8568437: Midgut, 24h, Low Virus, rep2; Aedes aegypti; RNA-Seq
library_construction_protocol: Midguts were dissected in 1X phosphate-buffered saline (PBS). Individual midguts were immediately homogenized for 30 sec at 6,000 rpm in tubes containing ~20 1-mm glass beads (BioSpec) in 500 μL of TRIzol (Life Technologies) and stored at -80°C. 100 μL of chloroform (Sigma-Aldrich) were added samples homogeneized by manual shaking for 30 sec. After a 5-min incubation at 4°C, samples were centrifuged at 4°C for 15 min at 14,000 rpm. The upper aqueous phase was harvested and transferred to a cold tube containing 400 μL of 2-propanol (Sigma-Aldrich). Samples were incubated at -20°C overnight and centrifuged at 4°C for 15 min at 14,000 rpm to pellet RNA. The pellet was washed twice with 1mL of 70% ice-cold ethanol (Sigma-Aldrich) at 4°C for 10 min at 14,000 rpm, and allowed to dry for 10 min at room temperature. Total RNA was resuspended in 7 μL RNase-free water. Directional libraries were prepared using the Smarter Stranded Total RNA-Seq kit- Pico Input Mammalian following manufacturer's instructions (Clontech, 635005). The quality of all libraries was assessed with the DNA-1000 kit (Agilent) on a 2100 Bioanalyzer and quantification was performed with Quant-It assays on a Qubit 3.0 fluorometer (Invitrogen). Clusters were generated for the resulting libraries, with Illumina HiSeq SR Cluster Kit v4 reagents. Sequencing was performed with the Illumina HiSeq 2500 system and HiSeq SBS kit v4 reagents. Runs were carried out over 65 cycles, including seven indexing cycles, to obtain 65-bp single-end reads. Sequencing data were processed with Illumina Pipeline software (Casava version 1.9).