it to the annealed RNA and incubated at 40°C for 2 hours, followed by 65°C for 15 minutes. We prepared the cRNA transcription master mix and added it to the cDNA and incubated at 40°C for 2 hours protected from light. We purified the labeled cRNA using Qiagen’s RNeasy 96-well columns (Qiagen, 74181) by adding 350ul of Qiagen RLT (without BME) to the cRNA followed by the addition of 250ul of 95% ethanol before applying to the plate column. After a 4 minute spin at 6000RPM, we washed the columns 3 times with 800ul buffer RPE. We dried the columns by spinning for 10 minutes and eluted the cRNA with 50ul of water. We measured the cRNA yield and dye incorporation using the Nanodrop 8000 Microarray measurement setting. We mixed 600ng of cRNA with blocking agent and fragmentation buffer (Agilent, 5190-0404) and fragmented for 30 minutes in the dark at 60°C. We added 2x Hybridization Buffer to each sample and loaded 40ul onto an 8-pack Hybridization gasket. We placed the microarray slides on top, sealed in the Hybridization Chamber, and incubated for 18 hours at 65°C. We washed the slides for 1 minute in room temperature GE Wash Buffer 1
