To detect the methylation status of the EphA5 promoter, six isolated colonies from each plate were picked, sequenced and analyzed using an ABI 3730 DNA Sequencer (Applied Biosystems).
Representative sequences of randomly picked 19-mer 4-base RNA oligonucleotides with free energy of self-hybridization ΔG 37°C of 2 kCal mol-1 or higher (2,768 sequences total, selected from a pool of 339,612 randomly picked sequences) are shown.
Recently, Tonkin et al. [ 16] demonstrated that even randomly picked sequences, which follow the basic rules for transit peptides (see above), could function as targeting sequences in apicomplexa, indicating a low complexity of transit peptides.
Compared to the randomly picked sequences with a GC content around 43%, the sequences close to the mGing locations only have a lower GC content, especially the 100 bp upstream and downstream of the mGing location.
For the hamster and deer mouse, because the definite locus information was lacking, we picked sequences that were as different from one another as possible (two for hamster, and three for deer mouse).
Scanning the upstream regions of genes in the Meta-UP dataset resulted in the identification of 32 transcription factors (TFs) with significantly enriched numbers of binding sites when compared to the distribution of transcription factor binding sites found in randomly picked sequences (chi2 test).
To circumvent PCR-induced artifacts Krsticevic et al. (2010) sequenced more than 100 clones and only picked gene sequence variants occurring twice or more.
At least 8 clones from each sample were picked for sequencing using BigDye sequencing kit and analyzed on an ABI 377 DNA Sequencer (Applied Biosystems, Foster City, CA).
Clones were randomly picked and sequenced until library sequencing is saturated; low probability of discovering a new mRNA reached due to the redundancy effect.
A representative and randomly picked protein sequence for each cluster was manually annotated by PSI-BLAST [ 35] and inspected for conserved domains using the NCBI conserved domain database.
