Transcriptome Profiling of Regenerative Tissue

Samples for high-throughput sequencing were prepared as previously described [12 (link)]. From each regenerating animal, we excised the region of the injury gap (∼3–4 mm wide) plus ∼3 mm of stump (‘old’) tissue on either side of the injury plane. The wet weight of an individual tissue sample was around 10–15 mg. Tissue samples of comparable size and weight were also excised from uninjured animals. During tissue sampling, every effort was made to separate the radial nerve cord from surrounding tissues. However, isolation of the pure nerve cord by surgical means turned out to be practically impossible. Therefore, our tissue samples also consistently contained small amounts of the surrounding connective tissue, an accompanying segment of the water-vascular canal and a stretch of the contractile coelomic epithelium of the body wall because of close anatomical proximity of these structures to the radial nerve cord (Figure 2). For the 454 platform, we generated three non-normalized libraries representing uninjured animals (38 individuals), days 2 and 6 post-injury (63 and 71 animals, respectively), and days 12 and 20 post-injury (62 and 66 animals, respectively). In addition, equal quantities of the above samples were combined to prepare a normalized library. The samples extracted from the regenerating animals on day 6 post-injury were only used for 454 sequencing to increase transcript diversity in the final assembled transcriptome, and were not subjected to sequencing on the Illumina platform (see below). Total RNA was extracted using TRI reagent (Sigma), assessed for quality on an Agilent 2100 Bioanalyzer with the RNA 6000 Nano chips, and subjected to two rounds of poly(A) selection using Poly(A)Purist technology (Ambion). Normalization procedure was performed with a TRIMMER kit (Evrogen) following the manufacturer’s protocol. The normalized cDNA was amplified using Advantage 2 Polymerase Mix (Clontech).
For Illumina sequencing two non-normalized libraries were prepared for each of the four conditions: (i) uninjured radial organ complex (total RNA samples were pooled from 4 and 3 animals for the first and second libraries, respectively); (ii) day 2 post-injury (20 and 19 animals were used); (iii) day 12 post-injury (20 animals were used for each of the libraries); and (iv) day 20 post-injury (15 animals were used for each of the libraries). The final stages in library preparation and sequencing were performed by sequencing service providers at the DNA Facility of the University of Iowa (Genome Sequencer FLX System, Roche) and the Genome Sequencing and Analysis Core Facility of the Duke Institute for Genome Sciences and Policy (Illumina Genome Analyzer IIx, Illumina). Raw sequencing reads from both the 454 and Illumina platforms were deposited at NCBI Sequence Read Archive (SRA) under accession number NCBI:SRA051990 [61 ].