An EST-based genome scan using 454 sequencing in the marine snail Littorina saxatilis: H ecotype
Total RNA was extracted from whole snails using TRIzol(R) reagent (Invitrogen) according to the manufacturer’s protocol. RNA quality and quantity were measured using the RNA 6000 Nano Chip Kit with an Agilent 2100 Bioanalyzer (Agilent Technologies). All the samples were standardized to 200 ng mkl-1 and equal volumes of the samples were combined into a pool (30 individuals). The pool was DNase-treated with Turbo DNA-free (Ambion) and purified with RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions; RNA quality and quantity was determined again at the end of the process. The RNA pool was used for double-strand cDNA synthesis using SMART technology (Zhu et al., 2001; BD Biosciences Clontech), and then the cDNA was purified using the QIAquick PCR Purification Kit (QIAGEN). Full-length cDNA was normalized using the DSN (duplex-specific nuclease; Shagin et al., 2002) normalization method (Zhulidov et al., 2004). cDNA synthesis and normalization were performed by Evrogen (http://www.evrogen.com, Moscow, Russia). The normalized cDNA pool was sequenced in one-half of a PicoTiterPlateTM using the Genome Sequencer FLX System (454 Life Sciences).