title: RNA-Seq of Rhinella marina: 3 adult female spleens (LPS 1)
design: 27 Adult Rhinella marina were subjected to an immune challenge with injections of either saline or LPS, with one LPS treatment being given a trans-dermal application of corticosterone (9 toads were assigned to each treatment). One hour prior to injections, toads in control and LPS-only treatments were given an application of peanut oil, while toads in the corticosterone treatment were given application of corticosterone dissolved in peanut oil. One hour post-injection of either saline or LPS, the toads were given the same trans-dermal applications. One hour following the second application, the toads were sacrificed and spleens from the toads were obtained, with spleens from each treatment being pooled together in groups of 3 and male and female spleens being pooled together (resulting in three samples per each treatment), and flash-frozen in liquid nitrogen. The samples were stored at -80 degrees Celsius to await RNA extraction. Grouped spleens were removed from -80 and homogenized into a powder using chilled mortars and pestles in liquid nitrogen. Powdered spleens were then transferred into Eppendorf tubes (one tube per group). Samples were resuspended with 900  l of QIAzol Lysis Reagent, and mixed gently using a 1mL syringe and a 21G   needle until a homogeneous solution was obtained. RNA was extracted from each of these grouped samples using a Qiagen RNeasy Plus Universal Mini Kit, which enriches for mRNA based on size. Aliquots of resulting extracted mRNA samples were sent to the Hudson Alpha Institute for Biotechnology in Huntsville, Alabama for paired-end RNA-seq using an Illumina HiSeq 2500 sequencer.