Trimmer direct kit

For 454 sequencing cDNA libraries generated from active, dehydration, inactive tun and rehydration stages were normalized using the duplex specific nuclease method (Trimmer-direct kit, Evrogen, Moscow, Russia), following manufacturers' recommendations [92] (link). The separately normalized cDNA libraries were pooled and sequenced using FLX Titanium chemistry at GATC Biotech. PolyA trimming and adaptor removal was performed using SnoWhite as for the Sanger data. Sequence reads have been deposited to the NCBI sequence read archive (SRA) under the study accession number SRA020123.

To maximize the discovery of C.hongkongensis genes, a normalized cDNA library was constructed. RNA from each sample was combined into a single pool and mixed well (see detail in S1 Table).The SMART (Switching Mechanism At 5’ end of RNA Template) kit (BD Clonetech, Mountain View, CA) was used to retrotranscribe total poly-adenylated RNA. First-strand cDNA was synthesized with SMART Oligo Ⅱoligonucleotide (5’-AAGCAGTGGTATCAACGCAGAGTACGGGGG-3’) and a modified oligo-dT primer (5’-AAGCAGTGGTATCAACGCAGAGTACTTTTGTTTTTTTTTCTTTTTTTTTTVN- 3’). Double-strand cDNA was obtained from 1μl of the first-strand PCR reaction with SMART PCR primer (5’-AAGCAGTGGTATCAACGCAGAGT-3’). Then, the amplified dsDNA products were purified using the QIAquick PCR Purification Kit (Qiagen, USA) and normalized using the Trimmer-Direct Kit (Evrogen, Moscow). The quality of the normalized cDNA library was determined on a 1.4% agarose gel. Approximately 2.5 μg of the normalized cDNA pool was used for a titration run using one quarter of a plate on the Roche 454 GS FLX sequencer (Roche, Basel, Switzerland), and then 15 μg of cDNA was sequenced with a whole-plate run on the same equipment at the University of Illinois, USA. The reads from this sequencing were deposited in the NIH Short Read Archive database with Run accession number SRR949615.

cDNA library preparation, normalization, and sequencing were performed at the Roy J. Carver Biotechnology Center, University of Illinois, Urbana-Champaign, following the procedures described by Lambert et al. [51 (link)]. Firstly, messenger RNA was isolated from 20 μg of total RNA using Oligotex kit (Qiagen, Valencia, CA), followed by the cDNA library construction with titanium adaptors (454 Life Sciences, Branford, CT). Secondly, the cDNA library was normalized using the Trimmer Direct Kit (Evrogen, Russia). Finally, this normalized library was sequenced on the 454 Genome Sequencer FLX+ system according to the manufacturer's instructions (454 Life Sciences, Branford, CT).