Total RNA was extracted using Trizol procedure, followed by mRNA isolation using an Oligotex mRNA Kit (Qiagen, Valencia, CA).
Label
Cy3 or Cy5
Label protocol
600ng adult fly poly(A)+ RNA was labeled with Cy3 or Cy5 random nonamer primer oligonucleotides (TriLink Biotechnologies, San Diego, CA) by reversed transcription in the presence of SuperScript II Reverse Transcriptase (Invitrogen). The labeled sample was purified with Chroma Spin column TE-10 (Clontech, Mountain View, CA).
Hybridization protocol
CPK6 50mer fiducial oligo control and hybridization buffer were added, and samples were applied to microarrays enclosed in custome-made chambers at 42C for 16-20 hours. Slides were then washed according to the protocol described by Nimblegen (Madison, WI) and dried by centrifugation.
Scan protocol
Scanned on an Axon GenePix 4000B scanner. Data were extracted from the scanned images using the NimbleScan 2.1 program (NimbleGen Systems, Inc.).
