title: DJF Pig oligo 27K1 ver1
status: Public on Feb 20 2008
contact.city: Tjele
contact.first: Jakob
contact.last: Hedegaard
contact.email: Jakob.Hedegaard@ki.au.dk
contact.address: PO-box 50
contact.department: Department of Genetics and Biotechnology
contact.country: Denmark
contact.institute: Aarhus University, Faculty of Agricultural Sciences
contact.phone: (+45)89991363
organism: Sus scrofa
manufacture_protocol: DJF_PIG_OLIGO_27K1_ver1 consists of 27.648 features - 25.210 unique oligonucleotides representing porcine transcripts and 2.438 control features - all printed as single spots in a 4 x 12 array with sub-arrays each consisting of 24 x 24 spots. The primary sequence source used for designing the oligonucleotides representing porcine transcripts was generated in the Sino-Danish Pig Genome Sequencing Project including more than 0.8 million EST sequences (Gorodkin et al.: Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 ESTs. Genome Biology 2007, 8:R45) and 3.8 million whole-genome shotgun sequences (Wernersson et al.: Pigs in sequence space: a 0.66X coverage pig genome survey based on shotgun sequencing. BMC.Genomics 2005, 6 (1)). An additional 1.6 million porcine sequences were retrieved from the public domains ENSEMBL (www.ensembl.org), NCBI (www.ncbi.nlm.nih.gov) and TIGR (www.tigr.org) yielding a total of approximately 6.2 million porcine sequences. A pipeline was set up to generate both candidate input sequences and final input sequences for oligonucleotide design. For human gene transcript, 15.700 sequences were generated and used as input for oligonucleotide design with SEPON (Hornshoj et al.: SEPON, a Selection and Evaluation Pipeline for OligoNucleotides based on ESTs with a non-target T-m algorithm for reducing cross-hybridization in microarray gene expression experiments. Bioinformatics 2004, 20 (3)). In addition, 5.500 transcript sequences for other mammalians and 5.100 sequences for uncharacterized genes were generated and used as input to OligoArray (Rouillard et al.: OligoArray: genome-scale oligonucleotide design for microarrays. Bioinformatics 2002, 18 (3)). The oligonucleotide design programs produced a total of 25.210 oligonucleotides representing approximately 19.000 genes. The oligonucleotides were synthesised by Isogen Life Science (www.isogen-lifescience.com) and delivered in 96 well plates. The oligonucleotides were resuspended in 0.01x SSC. Using a Sciclone ALH 3000 workstation (Caliper Life Sciences, www.caliperls.com), complete plates were rearrayed from 96 to 384 well library plates and incomplete plates were hitpicked into 384 well library plates. In average 200 pmol of each oligonucleotide were transferred to a 384 well spotting plate, dried by speed vac and resuspended in spotting solution (1 X Micro Spotting Solution Plus, Telechem International, www.arrayit.com). The 2.438 control features consist of 48 unique features printed in all 48 sub-arrays and 134 presumed blank features, which origin from presumed empty positions in the oligonucleotide plates supplied by Isogen Life Science. The 48 unique control features consists of a positive spot (also known as “landing lights”), a blank spot (spotting buffer), 10 oligonucleotides designed for Arabidopsis genes (Wang et al.: Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays. Genome Biology 2003, 4 (1) R5), 18 random 70 mer sequences selected to have no homology to known transcripts and 18 oligonucleotides representing the Lucidea Universal ScoreCard (GE Healthcare, www.gelifesciences.com). The positive spot was prepared by PCR amplification of a bovine EST fragment using a aa-dUTP containing dNTP mix followed by coupling of Alexa-555 or Alexa-647 using ARES DNA Labelling kits (Molecular Probes/Invitrogen, www.probes.invitrogen.com). The Arabidopsis and random oligonucleotides were synthesised by DNA Technology (www.dna-technology.dk). The 18 oligonucleotides representing the Lucidea Universal ScoreCard contains 10 oligonucleotides resulting in an intensity dilution series and 8 oligonucleotides resulting in different ratios when the labelling reactions are spiked in with the supplied RNA. The Lucidea Universal ScoreCard oligonucleotides were designed and synthesised by TIB MOLBIOL (www.tib-molbiol.com). The 46 unique control oligonucleotides were resuspended in 0.01x SSC and 200 pmol of each oligonucleotide was transferred to a 384 well spotting plate, dried by speed vac and resuspended in spotting solution (1 X Micro Spotting Solution Plus, Telechem International). The arrays were spotted on UltraGAPS slides (Corning Incorporated, www.corning.com) at 20 C/55 % RH using a ChipWriter Pro MicroArrayer (Bio-Rad Laboratories, www.bio-rad.com) with 48 x 946 MP2.5 pins (Telechem International, www.arrayit.com). Slides were dried and UV cross-linked (600 mJ) and stored in a vacuum desiccator until use.