array scanning and feature extraction protocol
P-AFFY-6 Affymetrix CEL analysis
20 ug of Biotinylated cRNA was fragmented and 10 ug hybridised to the Affymetrix Gene Chip Mouse Genome 430 2.0 array for 16 hours at 45C using an Affymetrix Gene Chip Fluidics Station 450 according to the manufacturers standard protocols.
Biotinylated cRNAs were then generated, using the GeneChip IVT labelling kit (Affymetrix).
Cells were washed twice with PBS, cultured for 3 h in ES medium + LIF without tet, trypsinized and re-plated onto new dishes. Cells were grown in medium deprived of tetracycline to perform miRNA induction and compared to cells grown in presence of tetracycline to switch off the miRNA expression (Parameters: start time = 18, time unit = hours, min temperature = 37, temperature unit = C, media = ES medium + LIF)
RNA extracts: 3 ug of total RNA were reverse transcribed to single stranded cDNA with a special oligo(dT)24 primer containing a T7 RNA promoter site, added 3' to the poly-T tract, prior to second strand synthesis (One cycle cDNA synthesis kit by Affymetrix).