title: Next generation sequence analysis of the transcriptome of Sydney rock oysters (Saccostrea glomerata) exposed to a range of environmental stressors
design: Sydney Rock oysters were obtained from a commercial hatchery in Port Stephens, NSW when they were approximately two years old and seven cm in length.Experimental treatments included hatchery controls, oysters deployed to field sites for 48 hours or 12 weeks, and oysters IP injected with LPS as a bacterial immunostimulant. Tissue from the gill and digestive gland of the oysters was homogenized by grinding with a mortar and pestel under liquid N2. RNA was extracted using the Trizol procedure (Invitrogen) followed by cleanup with the RNeasy kit (Qiagen). Qiagen’s oligotex kit and Invitrogen’s ribominus kit were used to remove rRNA. All pyrosequencing reactions were performed by the Ramaciotti Centre at the University of New South Wales.  Briefly, Libraries were prepared as described in "cDNA Rapid Library Preparation Method Manual - GS FLX Titanium Series - October 2009 (Rev. Jan 2010)", except for the following modifications: RNA was fragmented for 125seconds; enzymatic reactions and size selection were performed on the SpriWorks (Beckman Coulter).  Emulsion PCR (emPCR) titrations were carried out as described in "emPCR Method Manual - Lib-L SV - GS FLX Titanium Series - October 2009 (Rev. Jan 2010)."  Bulk emPCRs were carried out as described in "emPCR Method Manual- Lib-L LV - GS FLX Titanium Series - October 2009 (Rev. Jan 2010)." Sequencing was performed on a GS FLX (Roche) using the GS FLX Titanium Sequencing Kit XLR70 as described in "Sequencing Method Manual - GS FLX Titanium Series - October 2009 (Rev. November 2010)."  Image and signal processing were performed with Roche's software version 2.6.