title: SWEGENE human strand-specific cDNA array (cSense)
status: Public on Sep 01 2007
contact.city: Lund
contact.first: Johan
contact.last: Staaf
contact.address: Medicon Village
contact.country: Sweden
contact.institute: SCIBLU - Swegene Centre for Integrative Biology at Lund University
organism: Homo sapiens
description: Arrays contain strand specific cDNA probes. Probes are deposited as dsDNA and linked to the surface by NH2-modified ends. The deposited dsDNA is subsequently digested in situ in a strand-specific manner.
manufacture_protocol: Probe templates were amplified three times using different combinations of 5’-amino modified M13 universal sequencing primers. One reaction set used 5’-amino C-6 link modified M13 forward paired with non-modified reverse primer. A second reaction used 5’-amino C-6 link modified M13 reverse primer paired with unmodified forward primer. A third reaction used 5’-amino C-6 link modified primers in both orientations.
  After amplification, all PCR products were precipitated with ethanol and resuspended in phosphate buffer at approximately 100ng/µl for further deposition on arrays.
  Amino-modified PCR products were spotted onto CodeLink activated slides (Amersham) using a MicroGrid II array robot (BioRobotics).
  After printing, slides were coupled in a saturated NaCl chamber overnight at room temperature, blocked in 50mM ethanolamine, 0,1 MTris (pH 9) at 50degC 30 min followed by washes in distilled water and 4xSSC/0.1% SDS at 50degC.
  The deposited dsDNA was subsequently digested in situ in a strand-specific manner with T7 exonucelase 6. Slides were overlaid with 45ul exonuclease reaction mixture (1U/µl T7 exonuclease 6 in 1x reaction buffer, New England Biolabs), covered with a coverslip and left to incubate for 30 minutes at 25degC in a CMT Corning hybridization chamber. Following in situ digestion, probes were denatured by immersing the arrays in boiling water for two minutes.