PETRI-seq includes three parts—cell preparation, split-pool barcoding and library preparation. During cell preparation, cells are prepared for in situ reactions by fixation (formaldehyde) and permeabilization (lysozyme/lysostaphin). During split-pool barcoding, cells are split across 96-well plates three times for three rounds of barcoding by RT and two ligations. After barcoding, cells are lysed to release cDNA, which is subsequently prepared for paired-end Illumina sequencing. Each cDNA fragment in the library includes a UMI and three barcodes, which are all sequenced in read 1. The UMI is a sequence of seven degenerate nucleotides that can distinguish between unique transcripts and PCR duplicates. The three barcodes comprise a BC, which enables reads to be grouped by their cell of origin. In read 2, the cDNA is sequenced.