摘要Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues,providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression.In situ sequencing(ISS)is a targeted spatial transcriptomic technique,based on padlock probe and rolling circle amplification combined with next-generation sequencing chemistry,for highly multiplexed in situ gene expression profiling.Here,we present improved in situ sequencing(IISS)that exploits a new probing and barcoding approach,combined with advanced image analysis pipelines for high-resolution targeted spatial gene expression profiling.We develop an improved combinatorial probe anchor ligation chemistry using a 2-base encoding strategy for barcode interrogation.The new encoding strategy results in higher signal intensity as well as improved specificity for in situ sequencing,while maintaining a streamlined analysis pipeline for targeted spatial transcriptomics.We show that IISS can be applied to both fresh frozen tissue and formalin-fixed paraffin-embedded tissue sections for single-cell level spatial gene expression analysis,based on which the developmental trajectory and cell-cell communication networks can also be constructed.