BMC Genomics 2006, 7:154 http://www.biomedcentral.com/1471-2164/7/154 tail, which suggests that such ESTs might result from inter- nal oligo-dT priming during first-strand cDNA synthesis. BLASTX annotation of these 13,962 sequences identified a number of genes encoding proteins known to be impor- tant in the squid light organ for both development and symbiosis, such as: reflectin [26], actin [27], myeloperox- idase [28], aldehyde dehydrogenase [29] and nitric oxide synthase [30]. The identification of genes in the EST data- base known to play a significant role in the squid light organ serves as a proof-of-concept, suggesting that the additional sequences provided by the construction of the database will be invaluable for the identification of path- ways involved in the early development of the squid light organ and its colonization by V. fischeri. For example, this EST library has already played an integral role in the dis- covery of elements of the NF-kB pathway in the host squid [31]. Previous work on the E. scolopes – V. fischeri system has shown that the light organ is undergoing a rapid morpho- genesis in response to signals from both the external envi- ronment and colonizing V. fischeri in these early hours of the symbiosis (Fig. 2)[13]. The numbers of ESTs in each library as well as Gene Ontology (GO) descriptions of annotated ESTs are reflective of the active state of the organ during these early developmental stages. Specifi- cally, categories overrepresented included those involved with signal transduction, cell membrane, development and morphogenesis. Expression of genes in these catego- ries is not surprising in light of the fact that the host squid and the bacterial symbiont signal to one another through their cell surfaces, an activity that leads to the dramatic remodeling of host tissues. EST sequences often reflect the function of tissue from which the cDNA library was derived. To determine if the patterns of genes expressed in these cDNA libraries are unique to the squid light organ, we compared our results with that of the nonredundant EST database derived from the developing rat heart [17]. The squid light organ database is dominated by signal transduction (67%) and membrane-associated proteins (49%), whereas the database of the rat heart is dominated by cell growth and maintenance (63%) and intracellular activities (70%). Thus, in a broad sense, the content of these databases appear to mirror the physiological state of these organs. Conclusion This EST library is the first effort to generate a bioinfor- matics tool for the host side of the squid-vibrio associa- tion. This tool will allow us use this powerful binary model to ask specific questions to elucidate basic host responses to bacterial colonization in vivo, offering a complement to the more complex associations of verte- brates and the microbiota associated with their mucosa. Finally, the creation of a light organ-specific spotted microarray using this EST library will pave the way for the study of broad scale changes in host gene expression that underlie the dramatic developmental program of this symbiosis. Methods Animal maintenance and tissue preparation Adult E. scolopes and egg clutches produced by female adults were maintained at the Kewalo Marine Laboratory as previously described [32]. Approximately 1,500 light organs were collected in RNALater (Ambion) for the even- tual production of cDNA libraries from juvenile squid in five conditions: hatchling, 12-h aposymbiotic, 12-h sym- biotic, 48-h aposymbiotic, and 48-h symbiotic animals. All animals were hatched into Hawaiian offshore seawater (HOSW), which does not contain enough V. fischeri cells to result in light organ colonization, but does contain other bacteria (at 105-106 cells/ml) that naturally occur in the waters off of the Hawaiian island of Oahu. All light organs were dissected from animals, which had been anesthetized for ~2 min in 2% ethanol and HSOW, within 1 h before and after the time point specified for each con- dition, except for the hatchling condition, for which all dissections occurred within 1 h post-hatching. RNA isolation Total RNA was extracted from light organs in RNALater (Ambion) using Trizol reagent (Gibco BRL, Rockville, MD). Total RNA was quantitated by spectrophotometry and the quality was determined by 2% formaldehyde-aga- rose gel electrophoresis. Poly(A)+ RNA was isolated from total RNA samples using oligo-(dT)-cellulose chromatog- raphy. The method used for the construction of directionally cloned cDNA libraries [33,34] includes a column chroma- tography step that is aimed at eliminating unwanted DNA fragments (primers and adaptors) and short cDNAs (e.g., those consisting exclusively of poly(A) tail). Although aware of the possibility of excluding cDNAs derived from genuine short transcripts, this step has proven important to minimize generation of nuisance ESTs in large-scale sequencing projects. cDNA libraries DNase-treated poly-(A)+ samples were used to create 11 cDNA libraries: 5 start (non-normalized), 5 normalized and 1 subtracted. For each library, cDNA was primed with the following oligo (dT) primer, [TGTTACCATTCTGAT- GTTGGAGCGGCCGC-N[6–10]-T[18]]. Each primer con- tained a NotI restriction site for directional cloning and one of 11 unique library tags of 6 to 10 nucleotides (N [6– 10]), which identifies the condition of origin (Table 1) [35]. Double-stranded cDNA was ligated to EcoRI adap- Page 7 of 10 (page number not for citation purposes)