Background: Current commercial high-density oligonucleotide microarrays can hold millions of probe spots on a single microscopic glass slide and are ideal for studying the transcriptome of microbial genomes using a tiling probe design. This paper describes a comprehensive computational pipeline implemented specifically for designing tiling probe sets to study microbial transcriptome profiles. Results: The pipeline identifies every possible probe sequence from both forward and reverse-complement strands of all DNA sequences in the target genome including circular or linear chromosomes and plasmids. Final probe sequence lengths are adjusted based on the maximal oligonucleotide synthesis cycles and best isothermality allowed. Optimal probes are then selected in two stages - sequential and gap-filling. In the sequential stage, probes are selected from sequence windows tiled alongside the genome. In the gap-filling stage, additional probes are selected from the largest gaps between adjacen...