title: GSM2355585: Housefly_control rep1; Musca domestica; RNA-Seq
library_construction_protocol: At 48 h post-challenge, PBS- and virus-injected females were placed individually in tubes containing aliquots of 1 ml of Tri-Reagent (Sigma-Aldrich). Each sample was homogenized by adding ~ 20 zirconium beads (BioSpec Products, Bartlesville, OK) followed by vigorous shaking (30s) in a bead-homogenizer (FastPrep® Instrument, Qbiogene, Carlsbad, CA). Total RNAs were extracted according to the TriReagent protocol. Ethanol-precipitated RNA pellets were suspended in 100 µl DEPC-treated water and treated with RNase-free DNase (Qiagen). The isopropanol-precipitated total RNAs were re-extracted using SV Total RNA Isolation Kit (Promega, Fitchburg, WI, USA). RNA quantity and quality were assessed using a NanoDrop 2000 spectrophotometer  and the Agilent 2100 Bioanalyzer. Preparation and sequencing of RNA libraries were performed by ICBR/UF (Gainesville, FL USA) according to the manufacturer’s instructions (Illumina, Inc.) using the NextSeq500 platform. Briefly, the mRNA was enriched from 1 µg of total RNA per sample using oligo-dT attached magnetic beads and then subjected to thermal fragmentation using the elute, prime, and fragmentation mix from Illumina TruSeqTM v2 RNA sample preparation kit. RNA fragments were then converted to double-stranded (ds)-cDNA using reverse transcriptase and random primers provided in TruSeq RNA sample preparation kit. The ds- cDNA fragments were end-repaired by enzymatic polishing with T4 DNA polymerase and E. coli DNA polymerase I Klenow fragment. A single non-templated dA-tail was added to 3’-end of the repaired fragments and then ligated to NEB adaptors (NEBNext®Utra RNA library preparation kit). The required fragments were purified by AMPure beads (Agencourt; PN A63881) and enriched by PCR amplification. The amplified libraries were purified and quantified using the Agilent DNA high-sensitivity kit on an Agilent 2100 Bioanalyzer and qPCR.
attributes.tag: GEO Accession
attributes.value: GSM2355585