Protein Fisseq - a Targeted Fisseq Platform for in Situ Proteome Detection

Intact spatial information is key to answering lots of biological questions by bridging molecular information to biological functions and phenotypes. For example, to understand how neuron circuits function at the molecular level, we need to elucidate the ‘omic’ profiles of individual cells without disrupting cell-cell connections with in the circuits. The current high-throughput technologies, such as genome sequencing and protein mass spectrometry cannot maintain the spatial information necessary to achieve such single-cell resolution. Our lab recently developed the very first in situ sequencing technology termed RNA fluorescent in situ sequencing (RNA FISSEQ). It is capable to read unknown RNA sequences directly inside intact cells and tissues. Recently, we developed targeted FISSEQ platform for mRNAs and other RNA species such as sncRNAs, which allows detection and sequencing of a define subset of sequences of interest. Targeted FISSEQ can be readily adapted to detect DNA sequences. In light of this, I proposed to develop FISSEQ into an in situ proteomic technique termed “Protein FISSEQ”. Target proteins are identified by sequencing barcode oligos that are conjugated to antibodies. I show here that Protein FISSEQ is feasible by performing FISSEQ on barcoded alpha-tubulin antibody inside HEK293 cells. In addition, we are developing high-throughput methods to conjugate barcode oligos to a larger panel of antibodies as reagents for protein FISSEQ experiments.