Role of Bmi1 in Shh-mediated shift in gene expression levels

We investigated the role of Bmi1 in Shh-mediated shift in gene expression levels Primary cerebellar granule cell precursors (GCPs) cultures were prepared from P7 Bmi-1-/- or wild type mice as described before {Messer, 1977 #810}. Cells were grown in Dulbecco MEM high Glucose, supplemented with 10% FCS glutamine, 20 mM KCl and penicillin–streptomycin for 24 hours. Shh (R&D Systems) if appropriate was added for 24 hours to give at final concentration of 3 mg/ml. We performed 3 biological replicates for both WT and Bmi-1-/- cultures, and 2 biological replicates for both WT and Bmi-1-/- cultures, treated with Shh. Total RNA was isolated using RNeasy Mini Kit (Qiagen) and reverse-transcribed into double-stranded cDNA with One-Cycle cDNA Synthesis Kit (Affymetrix Inc., P/N 900431, Santa Clara, CA) and labelled biotin. Biotin-labeled cRNA samples were fragmented randomly to 35–200 bp and hybridized to GeneChip® Mouse Genome 430 2.0. An Affymetrix GeneChip Scanner 3000 (Affymetrix Inc., Santa Clara, CA) was used to measure the fluorescent intensity emitted by the labeled target.