SummaryTranscriptomic data and ChIP-seq data from bacteria are systematically analyzed and efficiently combined. We describe the software environment for analysis and the download and installation methods. Furthermore, we describe the analytical process and present the corresponding mini-test data, which can be conveniently restored and reproduced by users. Moreover, we provide the script for data consolidation, which allows multiple files to be rapidly merged. Overall, this protocol presents software parameters, R codes, and in-house Perl scripts for analyzing bacterial multi-omics data.For complete details on the use and execution of this protocol, please refer to Xin et al.