array scanning and feature extraction protocol
RNA was extracted for all time points samples and same amount of RNA from each time point were pooled together.
During labling after the purification of the cDNA, the samples were also pooled again before hybridisation. This enables that the same reference samples are used with each time point.
Place a coverslip in the hybridisation chamber face up. Place 50 µl of sample into each chamber on the cover slip. Place the array with the active side (labelled Agilent) down. When correctly positioned, the barcode should be facing up. Place the cover onto the chamber and tighten the screw as tightly as possible with your hand. Rotate the slide, check that the bubbles are moving and that each chamber is properly contained. Place in Agilent Hybridization oven and balance the rotor with an empty chamber. Hybridize at 65 C for 17 hours. Use Agilent wash buffers. Wash slide in wash buffer 1 for 1 min and in wash buffer 2 for 1 min. Rinse array in acetonitrile for 10 sec, and wash slide in Stabilization and drying solution for 30 sec.
nucleic acid labeling protocol
Fluorescently labelled cDNAs were prepared using the SuperScript(r) Direct cDNA Labelling System (Invitrogen). 5-10ug of total RNA (cleaned up 1x on QIAGEN RNeasy column and 2x on RNAeasy MinElute columns) in 6ul of water were mixed with 1ul of anchored Oligo(dT) Primer (2.5ug/ul) and 0.5ul of random hexamers, incubated at 70C for 10 minutes and cooled down on ice for 1 minute. The following reagents were added to each tube: 3ul of 5X First-Strand buffer, 1.5ul of 0.1M DTT, 0.5ul of dNTP Mix containing Alexa Fluor 555-aha-dUTP dCTP, 1.5ul of dNTP mix (0.5mM dATP,dGTP,dCTP; 0.3mM dTTP), 0.5ul of RNaseOUT(r) (40U/ul) and 1ul of SuperScript(r) III RT (400U/ul). The reactions were incubated at 46C for 3 hours in the dark. RNA was hydrolysed by addition of 7.5ul of 0.1M NaOH followed by incubation at 70C for 30 minutes. The solutions were then neutralised by addition of 7.5ul of 0.1M HCl. Alexa-555- and Alexa-647-labelled cDNAs were pooled and purified using a DNA purification module (Invitrogen). Labelled cDNAs were eluted in 25ul, and 5ul of Blocking Reagent (Agilent) and 25ul of 2x GEx hybridization buffer (Agilent) were added. The hybridization mixtures were heated at 100C for 5 minutes, cooled down and applied to DNA microarrays.
Cells were grown in EMM medium at 32C with or without caffeine and/or rapamycin and harvested from liquid cultures at OD 0.5.