title: Expression differences in the axolotl lateral line and ampullary  electrorecepotive organs as revealed by 454 sequencing
design: cDNA Synthesis and Normalization  For each RNA sample, two cDNA libraries were constructed using the Mint-Universal cDNA Synthesis Kit (Evrogen, Moscow, Russia), following the manufacturer’s Protocol II with the following modification: in place of the supplied CDS-3M adaptor, 3’ adaptors of our design were used. For the first cDNA library, the CDS-3M adaptor was replaced by CCG-Nor2-BaeI (5’-AAGCAGTGGTATCAACGCAGAGTGGCCGATACGCCCGTTTTTTTTTTTTTTTTTTTTVN-3’) and for the second cDNA library, the CDS-3M adaptor was replaced by CCG-Nor2-BpuEI (5’-AAGCAGTGGTATCAACGCAGAGTGGCCTTGAGGGCCTTTTTTTTTTTTTTTTTTTTVN-3’). These modified adaptors contain the recognition sites for the restriction enzymes BaeI and BpuEI, respectively, allowing for removal of the homopolymeric dA:dT region following cDNA normalization.  cDNA libraries were normalized using the Trimmer-Direct cDNA Normalization Kit (Evrogen) following the manufacturer’s protocol. Following normalization, libraries were restricted with either BaeI or BpuEI (New England BioLabs, Ipswich, MA) depending on which CCG-Nor2 3’ adaptor was used in a library’s cDNA synthesis. Small fragments were then removed from the libraries using Agencourt AMPure Beads (Beckman Coulter Genomics, Danvers, MA) using the manufacturer’s protocol. Libraries were then quantified by fluorometer using the TBS-380 Mini Fluorometer (Turner BioSystems, Sunnyvale, CA) with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA). Aliquots of both normalized libraries generated from the same tissue were pooled with 250 ng of each library in a final volume of 100 µl and used for 454 Sequencing.  454 pyrosequencing  For each normalized library set, 500 ng of normalized cDNA were prepared for sequencing using the GS FLX Titanium Rapid Library Preparation Kit and the GS FLX Titanium LV emPCR Kit (Lib-L) (Roche/454 Life Sciences, Branford, CT) according to the manufacturer’s instructions. Libraries were sequenced on the Genome Sequencer FLX System using the GS FLX Titanium Sequencing Kit XLR70 (Roche/454 Life Sciences). Low quality data were trimmed and excluded from the analysis. For assembly, GS FLX gsAssembler v. 2.6 was used and contigs were generated using a minimum overlap of 40 bases between individual reads that showed at least 95% identity.