title: Gryllodes sigillatus spermatophylax
design: Cricket rearing    Gryllodes sigillatus used in this study were descended from 500 adult crickets collected in Las     Cruces, New Mexico, in 2001, and used to initiate a laboratory culture maintained at a     population size of approximately 5000 crickets. Crickets, which were allowed to breed     panmictically, were housed in a 55 L plastic container in an environmental chamber maintained     at 32 ± 1 °C on a 14 h :10 h light/dark cycle.  Crickets were provisioned with Fluker’s cricket     chow (Fluker’s, Port Allen, LA, USA), water provided in 40 mL plastic tissue culture flasks     plugged with cotton dental rolls, and egg cartons to provide shelter and to increase the rearing     surface area.  Moistened peat moss provided in small plastic containers was made available both     as an oviposition substrate and as a source of additional water (11).    Accessory gland cDNA library preparation, sequencing and assembly    Five pairs of accessory glands were dissected from mature G. sigillatus males, immediately     frozen in liquid nitrogen and stored at -80 °C prior to RNA extraction.  Total RNA was isolated     using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s     instructions. Genomic DNA was removed by incubation with DNase (TURBO DNase,     Ambion, Austin, TX, USA) for 30 min at 37 °C.  Accessory gland RNA was further purified by     the RNeasy MinElute Clean up Kit (Qiagen, Valencia, CA, USA), again, following the     manufacturer’s protocol, and then eluted in 20 µl RNA storage solution (Ambion).  Full-length,     enriched cDNAs were generated from 2 µg total RNA using the SMARTer PCR cDNA synthesis     kit (BD Clontech, Mountain View, CA, USA). Reverse transcription was performed with the     SMARTScribe Reverse Transcriptase (BD Clontech) for 60 min at 42 °C and 90 min at 50 °C.      In order to prevent overrepresentation of the most common transcripts, the resulting double-    stranded cDNAs were normalized using the Kamchatka crab duplex-specific nuclease method     (12) of the trimmer-direct cDNA normalization kit (Evrogen, Moscow, Russia).  For 454     pyrosequencing, a cDNA aliquot of the accessory gland library was sent to the Advanced     Genomics facility at the University of Liverpool (http://www.liv.ac.uk/agf).  A single full-plate     run of 454 Titanium (Roche Applied Science) was performed using 3 µg of normalized cDNAs     processed by the ‘shotgun’ method.