The intestinal mucus layer forms an important barrier between the host epithelium and the gut microbiota. Goblet cells are responsible for producing mucus, and they can respond to microbial metabolites such as short-chain fatty acids through receptors including FFAR2. This study aimed to investigate whether goblet cell-specific deletion of Ffar2 affects the composition and functional activity of mucus-associated microbiota in the colon. RNA-seq data from wild-type and Ffar2 goblet cell-specific knockout mice were analyzed using a computational workflow. Reads were quality controlled and classified taxonomically to examine bacterial community composition. Alpha and beta diversity analyses were performed to compare bacterial diversity between groups. In addition, reads were mapped to a publicly available Helicobacter typhlonius reference genome to investigate bacterial gene expression, and mouse gene expression was also analyzed using the host read count matrix. Most RNA-seq reads were assigned to the mouse host, while the bacterial fraction was smaller and varied between samples. The bacterial community was dominated by a limited number of taxa, including Helicobacter, and H. typhlonius was detected as an important bacterial species in several samples. However, alpha diversity, beta diversity, and genus-level community profiles did not show clear or statistically significant differences between wild-type and knockout samples. Similarly, H. typhlonius gene expression and mouse host gene expression did not show significant genotype-associated differences after correction for multiple testing. The most informative bacterial result was the detection of highly expressed H. typhlonius genes in vivo, including genes related to stress response, metabolism, respiration, motility, and general cellular function. In conclusion, this study showed that bacterial RNA, especially from H. typhlonius, could be detected in mouse colon mucus-associated RNA-seq samples. However, no clear evidence was found for a strong effect of goblet cell-specific Ffar2 deletion on bacterial community composition, H. typhlonius gene expression, or mouse host gene expression in this dataset. The results suggest that individual sample variation and uneven bacterial read depth were major factors influencing the analysis.