title: WASHU_C.elegans_22.5K_V1
status: Public on Sep 14 2007
contact.city: St. Louis
contact.first: Elaine
contact.last: Mardis
contact.state: MO
contact.address: 4444 Forest Park Pkwy
contact.department: Genome Sequencing Center
contact.country: USA
contact.institute: Washington University
organism: Caenorhabditis elegans
manufacture_protocol: Long oligo probe design:
  An oligo selection algorithm program called TOP was modeled after PICK70 (http://se.osxgnu.org/pub/mirrors/sourceforge/arrayoligosel/). The algorithm utilizes FASTA input files in oligo selection. The oligo selection routine was designed to follow these steps: mask out any repetitive sequence elements or any highly similar portions of the input gene sequences using BLAST comparison, ensures all bases conform to A,C,G or T, checks that the defined oligo length conforms to a user defined Tm and G/C content, requires that the oligo conform to a user cutoff for homopolymer bases, assign secondary structure scores to oligos, and finally export oligos to a directory as text files (see supplementary material). The cumulative oligo scores assigned indicates the relative deviation of the oligo from the desired parameter. For genes that do not result in an oligo being selected we manually examine the input sequences and then if desired relax certain algorithmic parameters in order to select an oligo (see supplementary material for program details).
  Microarray slide production:
  Caenorhabditis elegans probes (oligos) were purchased from Illumina (http://www.illumina.com).  Oligonucleotides were solubilized at 50um in 3X SSC and 0.75 M betaine for printing.  The oligonucleotides were spotted onto epoxy coated glass slides (Corning) using a linear servo arrayer (P. Brown; Stanford University). The printed slides were placed in a 42C oven at 40% humidity for 12-14 hours.  After incubation, the slides were cross-linked using the UV Stratalinker 2400 (Stratagene) at 150 ujoules/cm2.  The slides were processed by submersing and shaking in a 0.2% SDS solution for 2 minutes, followed by three separate room temperature water baths, all at one minute per bath.  The final step was to place the slides in a 50 water bath for 20 minutes and then spin the slides dry.  The processed slides were placed in a light proof slide box and stored in a dessicant cabinet for future hybridization experiments.