NPAS2: An Analog of Clock Operative in the Mammalian Forebrain Martin Reick, Joseph A. Garcia, Carol Dudley, and Steven L. McKnight

Supplementary Material

Mouse BMAL1, PER1, PER2, CRY1 and GAPDH probes were obtained by RT-PCR from mouse brain RNA using the following primers: mBMAL1 forward: 5´-CATTCTCAGGGCAGCAGATGG-3´, mBMAL1 reverse: 5´-GGGCCACCTTCTCCAGAGGGC-3´; mPER1 forward: 5´-CTGCTACCTTCCCTTCTCC-3´; mPER1 reverse: 5´-CTCCTCAAGCTGCAGCAG; mPER2 forward: 5´-CCCACACTGGCTTCACCATGCC-3´; mPER2 reverse: 5´-GTGCTGCCTTCAGGCGCCTCC-3´; mCry1 forward: 5´-GGAAGCCTGCGGAAGCAGAGG-3´; mCry1 reverse: 5´-GGCACATCCATCTCTTCAATCAGG-3´; mGAPDH forward: 5´-ACCACAGTCCATGCCATCAC-3´, mGAPDH reverse: 5´-TCCACCACCCTGTTGCTGTA-3´. The probe for the 3´UTR of BMAL1 was obtained by RT-PCR with the following primers: forward: 5´-GTGCACAGAAGCATCATTGGTAGC-3´; reverse: 5´-CTGTCTAGTACATTATACTTTAACC. The resulting RT-PCR products were cloned into pGEMT and confirmed by DNA sequence analysis.

Probes for human PER1, PER2, CRY1 were obtained by PCR amplification from cDNA clones with these primers: hPER1 forward: 5´-GCTATCCACAAGAAGATTCTGCAGTTGGCG-3´; hPER1 reverse: 5´-GACAGCGCTGCTGACGGCGGATCTTTC-3´; hPER2 forward: 5´-GATGCCAGGTTTGTGGAGTTCCTG-3´; hPER2 reverse: 5´-GCTCTGTGAGCTCCTGAATGCTG-3´; hCry1 forward: 5´-ATGGGGTGAACGCCGTGCACTGG-3´; hCry1 reverse: 5´-CTGGCCACACTGCAGAGGATAAGCC-3´. For NPAS2 the KpnI fragment of the cDNA as used as probe. The probe for the Homo sapiens proteasome 26S subunit corresponds to bases 859-1279 of its cDNA, GenBank accession BC001747.