title: BCM-Shaulsky-7744-v1
status: Public on Aug 25 2006
contact.city: Houston
contact.first: Ezgi
contact.last: Booth
contact.email: eo135456@bcm.tmc.edu
contact.state: TX
contact.address: S430, One Baylor Plaza
contact.country: USA
contact.institute: Baylor College of Medicine
organism: Dictyostelium discoideum
manufacture_protocol: •	General array design:  Glass slides were coated with 3-Glycidoxypropyltrimethoxysilane (Aldrich).  Target DNA was printed on the activated glass slides on 200 µm centers with a Cartesian Pixsys5500 robot and with a GeneMachines OmniGrid Accent robot with Chipmaker II pins (Tele-chem Intl.).  The full ‘Cartesian’ array has 8x4 sub-arrays, each containing 22x22 targets (total 7,744 targets) as indicated in supplemental table 4 (see supplemental file ‘5_arrayMap1(table4).xls’).  The full ‘Accent” array has 22x24 targets (total 8,488 targets) targets.
  •	The source of the reporter molecules: 5,655 cDNA clones were from the Dictyostelium cDNA project; 987 extra cDNA clones were selected from a low redundancy screen of a lambda library and a plasmid library of late developmental stage, vegetative and early developmental stage of AX4 cells, respectively; 647 genomic DNA clones were selected as long open reading frames that matched published protein sequences; and 96 clones were from miscellaneous sources.  Sequences were compared to public databases and annotated 4.  The degree of redundancy is less than 15%.  The control targets that were made from the Dictyostelium ribosomal 17S RNA gene, histone H1, actin8 and mhcK as well some yeast genes and ‘no DNA’ controls.
  •	The method of reporter preparation:  DNA targets were amplified from plasmids by PCR with common oligonucleotides and their size verified by gel electrophoresis.  PCR products were purified by precipitation with 50% isopropyl alcohol 0.3 M sodium acetate pH5.2, washed once with 70% ethanol, dissolved in water and adjusted to 800 mM sodium chloride 200 mM sodium phosphate pH10.5.
  •	The spotting protocols used, including the array substrate, the spotting buffer, and any post-printing processing, including cross-linking:  Glass slides (Gold Seal Products, VWR) were washed by sonication in acetone for 10 minutes, rinsed twice in distilled water, immersed in 1 N NaOH for 10 minutes, washed in distilled water, and immersed for 3 minutes in 3% (v/v) 3-Glycidoxypropyltrimethoxysilane (Aldrich) made in 95% ethanol/acetic acid pH 5.0.  The slides were washed in 100% ethanol, dried with nitrogen gas and baked for at least 2 hrs at 100°C.  Target DNA in 800 mM sodium chloride 200 mM sodium phosphate pH10.5 solution was printed on the activated glass slides. Arrays were stored desiccated in the dark.
  •	Any additional treatment performed prior to hybridization: The arrays were treated with hot water for 1 min at 100°C for target denaturation prior to hybridization.
  For reference see Van Driessche, N., C. Shaw, et al. (2002). "A transcriptional profile of multicellular development in Dictyostelium discoideum" Development 129(7):1543-52.