Data provided in raw bead level format. Imported into R (http://www.r-project.org), background corrected (RMA algorithm) and summarised via the beadarray package for Bioconductor (http://www.bioconductor.org). Transcriptional profiles, summarised and background corrected as described, were log2 transformed and quantile normalised using the limma package for Bioconductor (http://www.bioconductor.org).
Illumina BeadScanner/BeadStation. Microarray Resources Centre, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.
Labelled cRNA was hybridised to Illumina Mouse WG-6 V2 BeadArrays using the manufacturer's standard protocol (http://www.illumina.com/). Performed at: Microarray Resources Centre, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.
RNA was isolated from the samples and cleaned up subsequently with Qiagen RNeasy micro kit, according to the manufacturer's instructions. The samples were quantified on Nanodrop (Thermo Scientific) and analyzed on Bioanalyzer (Agilent Technologies) prior to amplification and cRNA generation for hybridization onto Illumina BeadA.
Extracts were amplified and biotin labelled according to manufacturer (Illumina) standard protocol. Performed at: Microarray Resources Centre, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.
The embryonal carcinoma cell line P19 (P19EC) were cultured in DMEM, 10% fetal calf serum, mM L-glutamine (Gibco) and 50 ug/ml penicillin/streptomycin (Gibco).
Transfections of plasmids encoding either Blimp1-EGFP fusion protein or EGFP alone, were performed with Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. At 24 and 48 hours post transfection, cells were dissociated from the culture plates and subjected to flow cytometric analysis and sorting. At 24 hours EGFP or Blimp1EGFP positive cells were collected from 2 gates representing two levels of fluorescence, designated as high (HI) and low (LO). At 48 hours cells were collected from a single gate of cells showing fluorescence. A total of 2.5-5*10^5 cells were collected from each gate. The cells were then resuspended in Trizol (Invitrogen) and stored for 2-4 weeks at -80degC.