High purity dimethyl sulphoxide

Each pooled RNA sample was amplified (TargetAmp™ 1-Round Aminoallyl-aRNA Amplification Kit, Epicentre Technologies Corporation, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Following quality control (Nanodrop quantification and agarose gel electrophoresis) each aRNA sample was indirectly labelled and purified. Briefly, Cy dye suspensions (Cy3 and Cy5) in sufficient quantity for all labelling reactions were prepared by adding 40 μL high purity dimethyl sulphoxide (Stratagene, Hogehilweg, The Netherlands) per tube of Cy dye (PA23001 or PA25001; GE HealthCare, Little Chalfont, Bucks, UK). Each sample (2.5 μg aRNA) was denatured at 75°C for 5 min and then 3 μL 0.5 M NaHCO₃ pH8.5 and 1.5 μL Cy3 or 1.0 μL Cy5 dye was added achieving a total volume of 15 μL per reaction. Samples were incubated for an hour at 25°C in the dark, purified using Illustra AutoSeq G-50 Dye Terminator Removal Kit (Qiagen GE Healthcare) and concentration, dye incorporation and purity were assessed via spectrophotometer (NanoDrop) with products also visualised on a fluorescent scanner (Typhoon Trio, GE Healthcare).

Each pooled RNA sample was amplified (TargetAmp 1-Round Aminoallyl-aRNA Amplification Kit, Epicentre Technologies Corporation, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Following quality control (Nanodrop quantification and agarose gel electrophoresis) each aRNA sample was indirectly labelled and purified. Briefly, Cy dye suspensions (Cy3 and Cy5) in sufficient quantity for all labelling reactions were prepared by adding 42 μL high purity dimethyl sulphoxide (Stratagene, Hogehilweg, The Netherlands) per tube of Cy dye (PA23001 or PA25001; GE HealthCare, Little Chalfont, Bucks, UK). Individual samples (2.5 μg aRNA in 10.5 μL H₂O) were denatured at 75 °C for 5 min and then 3 μL 0.5 M NaHCO₃ pH 8.5 and 1.5 μL Cy3 dye added. The reference pool consisted of the same proportions per sample, but 1 μL Cy5 dye was used to label 2.5 μg pooled aRNA. Samples were incubated for an hour at 25 °C in the dark, purified using an Illustra AutoSeq G-50 Dye Terminator Removal Kit (Qiagen GE Healthcare), and concentration, dye incorporation and purity were assessed via spectrophotometer (NanoDrop) with products also visualised on a fluorescent scanner (Typhoon Trio, GE Healthcare).

Each pooled RNA sample was amplified (TargetAmp^TM 1-Round Aminoallyl-aRNA Amplification Kit, Epicentre Technologies Corporation, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Following QC (Nanodrop quantification and agarose gel electrophoresis) a reference (pool) sample was created by combining an equal amount of aRNA from each of the 144 reactions. Each aRNA sample was indirectly labelled (Cy3) and purified, while a similar (Cy5) labeling was undertaken for aliquots of the pooled reference sample. Briefly, Cy dye suspensions (Cy3 and Cy5) were prepared by adding 44 μL high purity dimethyl sulphoxide (Stratagene, Hogehilweg, The Netherlands) per tube of Cy dye (PA23001 or PA25001; GE HealthCare, Little Chalfont, Bucks, UK). Each aRNA (2.5 μg) was denatured at 70 °C for 2 min in 10.5 μL water and then 3 μL 0.5 M NaHCO₃ pH8.5 and 1.5 μL Cy dye (Cy3 or Cy5) was added and gently mixed. The suspension was incubated for 1 h at 25 °C in the dark and the excess label was removed by spin-column purification (Qiagen GE Healthcare). Dye incorporation and purity were assessed via spectrophotometer (NanoDrop) and, following agarose gel (1 %) electrophoresis, aliquots of the labelled aRNA were also visualised on a fluorescent scanner (Typhoon Trio, GE Healthcare).