Simultaneous single cell measurements of intranuclear proteins and gene expression

Abstract

Identifying gene regulatory targets of nuclear proteins in tissues remains a challenge. Here we describe intranuclear Cellular Indexing of Transcriptomes and Epitopes (inCITE-seq), a scalable method for measuring multiplexed intranuclear protein levels and the transcriptome in parallel in thousands of cells, enabling joint analysis of TF levels and gene expression in vivo. We apply inCITE-seq to characterize cell state-related changes upon pharmacological induction of neuronal activity in the mouse brain. Modeling gene expression as a linear combination of quantitative protein levels revealed the genome-wide effect of each TF and recovered known targets. Cell type-specific genes associated with each TF were co-expressed as distinct modules that each corresponded to positive or negative TF levels, showing that our approach can disentangle relative contributions of TFs to gene expression and add interpretability to gene networks. InCITE-seq can illuminate how combinations of nuclear proteins shape gene expression in native tissue contexts, with direct applications to solid or frozen tissues and clinical specimens.

Single-cell RNA-sequencing reveals widespread personalized, context-specific gene expression regulation in immune cells

“…the functional proportion). 49,50 Consequently, it can be difficult to define the direct causal factor solely using co-expression QTL analysis. Nevertheless, we expected that by taking a pathway-level view, the downstream genes of transcription factors would have a high correlation with the functional protein level of the transcription factor and would be more easily picked up than a single gene.…”

Single-cell multi-omic analysis of thymocyte development reveals drivers of CD4/CD8 lineage commitment

“…While differences between sequencing-based and fluorescence-based protein measurements such as noise (e.g., spectral overlap) and sensitivity (e.g., barcode amplification) might limit the direct translation of gate position, large CITE-seq panels could provide a useful platform for screening potential combinations of fluorescence-based markers. The CITEseq method is currently limited to the measurement of surface proteins (Stoeckius et al, 2017), but development of emerging methods such as inCITE-seq (Chung et al, 2021) to facilitate the simultaneous measurement of RNA, surface proteins, and large panels of intracellular proteins could greatly enhance the ability to generate hypotheses about molecular pathway activity, gene regulatory networks, and transcription and translation dynamics. Furthermore, since thymocytes actively traverse the thymic cortex and medulla over the course of their development, imaging could provide a valuable dimension to our current understanding of the thymocyte developmental timeline (Germain et al, 2012).…”

NEAT-seq: Simultaneous profiling of intra-nuclear proteins, chromatin accessibility, and gene expression in single cells

“…Recently, this approach was extended to measuring intracellular proteins [4][5][6][7][8] . However, quantification of nuclear gene regulatory proteins along with chromatin accessibility profiling and RNA-seq has not been demonstrated.…”

“…While multiple groups have demonstrated sequencing-based surface protein quantification using barcoded antibodies 1,3,13,14 , application to nuclear proteins has been challenging due to high levels of background oligo-antibody staining in the nucleus, potentially driven by the highly negatively charged conjugated single stranded DNA (ssDNA) oligo 5,15 . One approach to reduce non-specific staining is to saturate cells with single stranded nucleic acids or other negatively charged polymers in an attempt to block cellular components that bind nonspecifically to ssDNA 5,6,8,15 . However, we hypothesized that directly blocking the charge of the antibody oligo might dramatically improve signal over background.…”