Highly multiplexed subcellular RNA sequencing in situ

Abstract

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

Fig. 1

Construction of 3D RNA-seq libraries in situ. After RT using random hexamers with an adapter sequence in fixed cells, the cDNA is amplified and cross-linked in situ. (A) A fluorescent probe is hybridized to the adapter sequence and imaged using confocal microscopy in human iPS cells (bar: 10 um) and fibroblasts (bar: 25 um). (B) FISSEQ can localize the total RNA transcriptome in mouse embryo and adult brain sections (bar: 1 mm), and whole-mount Drosophila embryos (bar: 5 um), although we have not sequenced these samples. (C) 3D rendering of gene-specific or adapter-specific probes hybridized to cDNA amplicons.

Fig. 2

Overcoming resolution limitations and enhancing the signal-to-noise ratio. (A) Ligation of fluorescent oligonucleotides occurs when the sequencing primer ends are perfectly complementary to the template. Extending sequencing primers by one or more bases, one can randomly sample amplicons at 1/4^th, 1/16^th, and 1/256^th of the original density in fibroblasts (bar: 5 um). (B) Rather than using an arbitrary intensity threshold, color sequences at each pixel are used to identify objects. For sequences of L bases, the error rate is approximately n/4^L per pixel, where n is the size of the reference. By removing unaligned pixels, the nuclear background noise is reduced in fibroblasts (bar: 20 um).

Fig. 3

Whole transcriptome in situ RNA-seq in primary fibroblasts. (A) From deconvolved confocal images, 27-base reads are aligned to the reference, and alignments are spatially clustered into objects. (B) 90.6% of the amplicons align to the annotated (+) strand. (C) mRNA and non-coding RNA comprise 43.6% and 6.9% of the amplicons. (D) GO term clustering for top 90 ranked genes. (E) 2,710 genes from fibroblast FISSEQ compared to RNA-seq for fibroblast, B-cell, and iPS cells. Pearson’s correlation is plotted as a function of the gene expression level. (F) Subcellular localization enrichment compared to the whole transcriptome distribution. (G) 481 amplicons map to the FN1 mRNA, showing an alternatively spliced transcript variant and a SNP.

Fig. 4

Functional analysis of fibroblasts during simulated wound healing. (A) In EGF media, ribosomal RNA comprises 81.6% of the amplicons. (B) 109,646 reads from EGF media compared to 14,960 reads from FBS media (different colors denote genes). (C) Top 100 ranked genes from FBS vs. EGF FISSEQ clustered for functional annotation. (D) An in vitro wound healing assay allows cells to migrate into the wound gap. The image is segmented based on the cell morphology. (E) 4,533 genes from migrating and contact inhibited cells are compared. (F) Twelve genes are differentially expressed (Fisher’s exact test p-value<0.05 and >5-fold; 180 genes). (G) Top 100 genes in fibroblasts are enriched for terms associated with ECM-receptor interaction and focal adhesion kinase complex (bold letters). During cell migration, genes involved in ECM-receptor-cytoskeleton signaling and remodeling are differentially expressed (red letters).