In situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved tissue. This protocol can be used to detect RNA molecules at the single cell level to aid in the identification of cell types according to their gene expression. The technique uses padlock probes to target desired genes of interest and rolling circle amplification to amplify signal for a high throughput methodolgy of spatial transcriptomics. With the use of barcode sequencing, identification of numerous genes is possible through multiplexing.Version 2 Update: Only minor mistakes were corrected for version 2 of this protocol, no major changes to protocol were done. Some references were also updated.