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Diverse immune populations reside in non-lymphoid organs and contribute to immune defense and tissue homeostasis. However, these cells are often hard to study due to low cell abundance and high heterogeneity. Recent advancements in single-cell sequencing technology provides a powerful high parameter tool to study these peripheral immune populations. But current single cell experiments are costly and limited by sample throughput. To address these limitations, we have developed a novel sample multiplexing approach for high throughput single-cell sequencing. In this study, we performed single-cell sequencing analysis of thousands of immune cells isolated from peripheral tissues in mice, and utilized a DNA barcoded universal antibody to sample multiplex up to 12 samples in a single experiment. This allowed us to combine samples from different mice and tissue types into a single pooled sample, significantly reducing experimental scale and cost, while eliminating potential batch effects. The sample pool was captured on the BD Rhapsody™ system and a targeted assay was performed to measure gene expression of ~400 genes. We were able to de-multiplex the pooled samples with high specificity after sequencing. The targeted gene panel provided robust clustering of the major immune cell types, enabling us to perform immuno-profiling and compare gene expression of major cell populations across different tissues. We observed distinct tissue-specific expression profiles of major immune populations, and further investigation of the differentially expressed genes may provide better understanding of the interactions between these tissue immune populations and their local environment.