title: Agilent-024708: Drosophila euchromatic probes, plus potential heterochromatin [Probe name version]
status: Public on Mar 06 2012
contact.city: Princeton
contact.first: Bing
contact.last: He
contact.email: bhe@princeton.edu
contact.state: NJ
contact.address: Washington Rd
contact.department: Molecular Biology
contact.country: USA
contact.institute: Princeton University
organism: Drosophila melanogaster
description: In order to acquire potential Drosophila heterochromatin sequences for microarray construction, DNA libraries were prepared from approximately 250 2L- and 2R- embryos collected at the cellular blastoderm or early gastrulation stages, before the heterochromatin is underreplicated. The embryos were dechorionated and digested with proteinase K prior to sodium acetate/ethanol precipitation. DNA was sheared on a Covaris S2 instrument to a mean length of 200 bp, end repaired and ligated to adaptors for Illumina sequencing. The resulting short sequence reads were mapped to the Release 5 reference Drosophila genome using BWA with default settings (Li and Durbin, 2010). Sequences that mapped to the euchromatic arms and the heterochromatin sequences contiguous with the euchromatic arms ( h ) were removed. The remaining sequences, which were considered probable unmapped heterochromatic sequence, were assembled into contigs using SSAKE (Warren et al. 2007).  The reads were first quality trimmed using SSAKE provided scripts to require at least 20 consecutive bases above a threshold value of 10. The SSAKE assembly was run using default parameters. Custom microarrays were designed for manufacture by Agilent. Assembled contigs of 60 nt length or greater following masking by DUST were passed to ArrayOligoSelector (http://arrayoligosel.sourceforge.net/) for probe design, using a database of all assembled contigs plus the Drosophila genome as the background set used to determine the maximally unique probe for each contig sequence (Bozdech et al., 2003). Probe sequences were supplied to Agilent for manufacture of custom microarrays, available as Agilent design ID 024708. About 80,000 60-mer nucleotide probes were selected corresponding to the assembled contigs, including both forward and reverse complements in order to allow for use in subsequent expression analysis (~ 20,000 reverse probes that were not expressed were later removed from the microarray design). The microarray also contains ~23,000 probes designed by Agilent for expression sequences (annotated genes) covering the entire reference genome as reference probes.
manufacture_protocol: see manufacturer's web site at http://www.agilent.com/