title: Agilent-020980 Acropora formosa coral 8x15K
status: Public on Mar 26 2014
contact.city: Vicksburg
contact.first: Kurt
contact.middle: A
contact.last: Gust
contact.email: kurt.a.gust@usace.army.mil
contact.state: MS
contact.address: 3909 Halls Ferry Rd.
contact.department: Environmental Laboratory
contact.country: USA
contact.institute: US Army ERDC
contact.phone: 601-634-3593
organism: Acropora formosa
description: Coral consortium including endosymbionts of the branched coral Acropora formosa.
  Microarray Development Methods: An RNA library was assembled by sampling RNA from controls and all RDX exposure treatments performed in the 5d A. formosa bioassay. Full-length cDNAs were reverse transcribed from the coral RNA library using Clontech SMART-PCR (Clontech Laboratories Inc., Mountain View, CA). In order to generate sequence information for as many unique cDNAs as possible, the cDNA library was normalized using the Trimmer cDNA Normalization Kit (Evrogen JSC, Moscow, Russia) to insure sequencing of both high- and low-abundance transcripts. The normalized cDNA library was sequenced using massively-parallel Roche 454 GS-FLX pyrosequencing (Margulies et al. 2005) with several modifications that improved sequence reproducibility while reducing labor-intensive sample manipulations (Wiley, et al. 2009 and Roossinck, et al. 2010). The conceptual pipeline for sequence processing and annotation has been illustrated in supplemental Figure S1. Sequences were quality filtered and sequence assembly was conducted using CAP3 software. Contigs and singletons were searched against the NCBI GenBank protein database using blastx where E ≤ 10-5 was considered a significant match to an expressed sequence tag (EST). Gene targets were annotated where possible with identity, functionality using KEGG and KOG databases. In order to study the metabolic profile of coral symbionts, sequence reads were split into two different bins using the phylogenetic information provided by BLAST. Sequences associated with non-coral species were extracted at the Anthozoa class. Metabolic profiles of coral and non-coral sequences were then compared by calculating the ratio of the transcripts. The 7,500 sequences identified with the lowest E values (which represent the best homology to known genes) were uploaded to eArray (Agilent Technologies) in both forward and reverse sequence orientation (due to lack of reading-frame information to assess the sense strand). eArray was utilized to incorporate these 15,000, 60mer oligonucleotide probes into a custom microarray design (Agilent Technologies) for A. formosa. This design represents 7,500 sense-antisense probe-pairs providing quality control of the microarray analysis for cross hybridization. One probe in each probe pair represents a nonsense sequence and our expectation was that no target should have specific binding to it (see above for anti-sense strand exclusion methods).
manufacture_protocol: See manufacturer's website (www.agilent.com).