What are the limitations of CITE-Seq?

CITE-Seq is a sequencing-based technique that combines single-cell RNA sequencing with cell surface protein analysis, offering a comprehensive view of both transcriptomes and proteomes within individual cells. Although this technique offers several advantages, it does come with a few limitations too. 

• CITE-seq is limited to tagging cell surface proteins. It cannot be used to tag epitopes inside a cell as that would entail puncturing the cell surface membrane, which would release the RNA content from within the cell, rendering the sample incompatible for use with any RNA sequencing technology.  
• The specificity with which antibodies bind to an epitope can impact the accuracy of CITE-seq results. 
• The presence of multiple antibodies increases the risk of cross-reactivity and inaccurate results. 
• Using enzymes that digest proteins to dissociate cells can alter surface protein levels, which may impact the accuracy of protein analysis. Additionally, the binding of antibodies to surface proteins might activate cellular signaling pathways, potentially altering the cell’s transcriptome. Fixing cells on ice and thoroughly testing the effects of cell dissociation and antibody binding on the specific tissue being studied may help mitigate these issues.