title: directional RNA-Seq of Bison bison: adult female spleen: Sample B423SP
design: "Tissue was harvested immediately after euthanasia. Approximately 100 mg of tissue was flash-frozen in liquid nitrogen and stored at -80 degrees Celsius until RNA was extracted. Frozen tissue was pulverized using a mortar and pestle, and total RNA was extracted using TRIzol RNA Isolation methods (Life Technologies). Quantitative and qualitative RNA assessments were made using Nanodrop (Thermo Scientific) and Bioanalyzer 2100 (Agilent ) using the RNA 6000 Nano chip. A 2-ug total RNA sample was suspended in 10 uL of DEPC-treated water.  Total RNA was depleted of rRNA and the libraries were prepared using the TruSeq stranded total RNA sample preparation kit for human/mouse/rat (Illumina). The sizes of the libraries were determined on a Bioanalyzer 2100 (Agilent) using the DNA1000 chip and quantifiedon aQubit 2.0 using the broad range DNA assay (Invitrogen). Libraries were diluted to 2 uM and pooled. The pooled libraries were quantified again, this time using the Qubit high sensitivity assay, to recheck concentrations.Prepared samples were loaded into six lanes on a high output flow cell ??at a concentration of 7 pM and cluster generation was performed usinga cBot (Illumina). The flowcell was runonan IlluminaHiSeq 2500 (2X100 High Output)."