Abstract
In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8,000 genes were detected with 7 × 104 unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions.
Highlights
In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions
We demonstrated that photo-isolation chemistry (PIC) with RNA-seq for tissue sections could detect the expression of approximately 10,000 genes from only a few dozen cells from the mouse neural tube
More than 10000 genes were detected from 100 cells by UV irradiation to intracellular and nuclear structures, thereby highlighting hundreds of transcripts localised in stress granules (SGs) and nuclear speckles (NSs)
Summary
Expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. We establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles from photo-irradiated regions of interest. PIC transcriptome analysis detects genes expressed in small distinct areas of the mouse embryo. PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions. By taking advantage of the fact that light can change molecular properties with a resolution up to the diffraction limit, we here introduce photo-isolation chemistry (PIC), which enables transcriptional profiles of photo-irradiated cells alone to be determined. PIC identifies genes uniquely expressed in spatially distinct areas with a resolution up to subcellular and subnuclear levels
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