title: Thalassiosira pseudonana NimbleGen 36mer oligo tiling array
status: Public on Jan 05 2008
contact.city: Seattle
contact.first: E.
contact.middle: Virginia
contact.last: Armbrust
contact.email: armbrust@uw.edu
contact.state: WA
contact.address: Ben Hall IRB, 616 NE Northlake Pl, Room 306
contact.department: Oceanography
contact.country: USA
contact.institute: University of Washington
organism: Thalassiosira pseudonana CCMP1335
description: The layout files for the individual chips that make up the 8-array set are linked below as supplementary files.
manufacture_protocol: 1.  Tiling array design: A total of 1,308,958 36-mer probes were chosen uniformly from both strands of the 34 Mb T. pseudonana genome (Version 2, http://www.jgi.doe.goc) with gaps of 10 nucleotides between consecutive probes. No probes were chosen from the ambiguous or repetitive regions of the chromosome (Todd Richmond; personal communication) or the unassembled scaffolds. The selected probes were synthesized on eight glass-based microarrays, with 178,600 probes on the first 7 microarrays and 58,758 probes on the eighth microarray. Each array also included ca. 500 random duplicated probes for normalization and noise estimation purposes. The location of the probes on each array was randomized in order to eliminate any positional bias in the synthesis and hybridization.
  2. Synthesis: Two complete sets of the tiling microarrays were synthesized for hybridization with labeled cDNA from the control and the low silicon growth conditions. Microarrays were fabricated using a modified Maskless Array Synthesizer (MAS) as described previously (Sing-Gasson et al. 1999, Nuwaysir et al. 2002) on SuperClean (ArrayIT) glass substrates functionalized with N-(3-Triethoxysylylpropyl)-4-hydroxy-butryamide (Gelest Inc). Delivery of standard DNA synthesis reagents (Proligo) and NPPOC phosphoramidite (Nimblegen) was performed via an Expedite DNA synthesizer.
  3. Array Processing: After synthesis, microarrays were deprotected by immersion in EDA/EtOH (1:1 vol) for two hours at room temperature.  Arrays were then washed in water, dried with argon, and stored desiccated prior to hybridization.  Hybridization was performed with 10ug of fragmented cRNA in 300ul in the presence of 100mM MES, 1M Na, 20mM EDTA and 0.01% tween20 for 17 hours at 45C.  After hybridization, arrays were washed with a non stringent wash buffer (NSWB) (6X SSPE and 0.01% Tween 20) followed by incubation with a stringent wash buffer (100mM MES, 0.1M Na+, 0.01% Tween 20) for 30 minutes at 45C.  Arrays were stained with streptavidin-cy3 (Amersham) for 25 minutes at room temperature, washed with NSWB, rinsed with Final Wash Buffer (Nimblegen) and dried under argon. Arrays were scanned using an Arrayworx scanner (Applied Precision).  Array feature intensity was extracted from images using in-house software.