title: [UCDavis]-[Delta smelt]-[18240]-[V:1.2]
status: Public on Aug 18 2010
contact.city: Davis
contact.first: Richard
contact.middle: Edward
contact.last: Connon
contact.email: reconnon@ucdavis.edu
contact.state: CA
contact.address: One Shields Avenue
contact.department: Anatomy, Physiology and Cell Biology
contact.country: USA
contact.institute: University of California at Davis
organism: Hypomesus transpacificus
description: ONLY CLONES OF INTEREST HAVE BEEN SEQUENCED AND IDENTIFIED.
manufacture_protocol: Delta smelt microarray with 18,240 features; comprised of 8448 cDNA fragments printed in dulpicate (16,896) and 1344 control spots. Specific details: We constructed a delta smelt microarray using 8448 PCR amplified fragments from a normalized cDNA library. A cDNA library was created by ligating expressed sequence tags (ESTs) into p-BS plasmid vectors and cloned into chemically competent Escherichia coli cells (BioS&T Inc, Montreal, Quebec, Canada). Products were PCR amplified from 1µl bacterial suspension, and visualized on agarose gels.  Purified PCR fragments ranging in size from 1- 4kb, along with control spots, were pin-printed on glass slides in a 20 x 19 block format, with 48 blocks per microarray.  Microarrays were printed at the Array Core facility at Robbins Hall, UC Davis (now closed down). Microarray control spots included a number of hybridization tags comprised of a pooled PCR product from all spots on the array, H. transpacificus DNA, and four Spot Report System of alien PCR products from Arabidopsis thaliana; CAB, RCA, RBCL and LPT4 (Stratagene, USA).  Blank control spots consisting of 1x Nexterion buffer solution were printed interspaced with the above controls and as the last 12 spots in each block, and used to assess printing quality and potential cross contamination resulting from printing. Only genes that were differentially expressed following esfenvalerate exposure were sequenced.  Aliquots of respective bacterial colonies used to create the microarray were sequenced using standard M13 forward and reverse primers.  Sequencing was carried out at the CA&ES Genomic Facility, UC Davis.  Basic Local Alignment Search Tool; translated nucleotide (BLASTx) searches were performed on specific fragments that responded significantly to the exposure treatments. Sequences were annotated according to homologies to protein database searches using translated nucleotide sequences and direct nucleotide queries (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Homologies to other fish species were of primary consideration on gene annotation. Sequences were only annotated if they were found to have a BLASTx match with the expect value smaller than 1x10-5 and a score above 50. Accession number and species’ match were recorded with each annotation.