Sequencing of the IRF-4 promoter
For analysis of the IRF-4 promoter region for permanent aberrations such as insertions/deletions or mutation, we PCR-amplified two fragments from genomic DNA, which was extracted from depicted cell lines with a commercial kit (Qiagen, Hilde, Germany) as recommended. The primers were 1-forward: 5′-TTGAGATGGAGTCTTGCTCTGT-3′, 1-reverse: 5′-CCAGGACCTCAGGAGGCCAGTCA-3′; 2-forward: 5′-AGCGGTGAAACTGAGAGTGCGAGGT-3′, 2-reverse: 5′-GCCACATCGCTGCAGTTTAG-3′. The products were cloned with the ‘TOPO TA cloning kit’ (Invitrogen, Groningen, The Netherlands). After bacterial amplification of the cloned PCR fragments by standard procedures, at least three clones from each sample were sequenced with an automated sequencer (ABI Prism 377, Applied Bio-systems, Foster City, USA) as recommended by the manufacturer.