In situ sequencing

For cells, the coverslip was mounted in a flow cell, and in situ sequencing reactions were performed using automated fluidics. For embryos, reactions were performed manually in a six-well plate. Samples were treated with calf intestinal phosphatase before the first primer hybridization and before each cleavage reaction. In situ sequencing was performed using sequencing-by-ligation chemistry. Samples were exchanged into an imaging buffer after each round of sequencing. Images were acquired using confocal microscopy. Immunostaining and immunofluorescence imaging were performed after in situ sequencing.