title: GU_rainbowtrout_15k_v3.0
status: Public on May 10 2012
contact.city: Gothenburg
contact.first: Marija
contact.last: Cvijovic
contact.address: Chalmers Tvärgata 3
contact.country: Sweden
contact.institute: Chalmers Univeristy of Technology
organism: Oncorhynchus mykiss
manufacture_protocol: An oligonucleotide microarray was designed for the Geniom platform, which is fully automated system with in situ synthesized probes (Febit, Heidelberg, Germany). Probes for all eelpout transcripts were generated in a two-stage process. First, a preemptive clustering step, where very similar transcripts were grouped into single entities, was used to avoid losing good probes due to presumed cross-hybridization to splice variants and isogenes. Next, probes were designed using OligoArray 2.1 which is an algorithm using thermodynamic approach  to calculate specificity and to predict secondary structures. The probe design were run using the following parameters: the probe length were set to 50 nucleotides, Tm-range to 90°–95°, GC-content to 45%–55%, folding temperature to 70° and cross-hybridization temperature to 50°. The default settings were kept for all other parameters and no specificity check was done between transcripts within the same cluster. For sequences where no satisfactory probe could be found, a second run with a slightly less strict GC-content (45%–60%) were done. Since massively parallel pyrosequencing is dependent on a fragmentation step, where the mRNA is randomly cut into processable pieces, the strand orientation is lost and thus the direction of transcription for the generated reads and, ultimately, for the assembled contigs. We therefore designed probes for all assembled transcripts and the corresponding reverse complements resulting in 39,618 (74%) and 39,733 (74%) probes respectively. In total, 117,316 probes were used and distributed over eight separate microarrays. Each array contained 15,648 spots, including controls, and were synthesized in situ on one single biochip using the 5' standard synthesis set for Geniom.