Construction of multiplexed transcriptome NGS libraries of microdissected tissue samples based on combinational DNA barcode microbeads

Abstract The combination of single‐cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)‐seq as a medium‐throughput, multiplexed approach combining Smart‐3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB‐seq to RNA extracts, fresh frozen (FF) and formalin‐fixed paraffin‐embedded (FFPE) mouse brain tissue samples. CDB‐seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost‐effective way, providing a promising method for future spatial transcriptomics.