Abstract
We present barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel insitu analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POLR2A, respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglial cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene–gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research.
Highlights
Spatial transcriptomics is a rapidly evolving field, with recent developments in multiplexed in situ technologies paving the way for spatial imaging of the genome and transcriptome at an unprecedented resolution [1]
Spatial transcriptomics is a rapidly evolving field and the interests of obtaining 3D information of transcripts in cells, tissues, and even whole organisms have sparked the development of a number of promising technologies, including but not limited to in situ padlock [6], Fluorescent In Situ Sequencing (FISSEQ) [5,45], ExSeq [28], MERFISH [10], seqFISH+ [12,13], slide-seq [46], HDST [47], INSTA-seq [48] and STARmap [14]
Compared with in situ padlock probe methods, BOLORAMIS removes the need for reverse transcription (RT) by using a RNA-splinted DNA ligase, reducing potential detection bias and experimental cost resulting from RT
Summary
Spatial transcriptomics is a rapidly evolving field, with recent developments in multiplexed in situ technologies paving the way for spatial imaging of the genome and transcriptome at an unprecedented resolution [1]. Spatial profiling of gene expression patterns in cells of a given tissue can provide intricate molecular maps, allowing quantification of transcripts and understanding of cellular function in a particular environment. We proposed the idea of Present address: Eswar P.R. Iyer, 10X Genomics, Pleasanton, CA 94588, USA.
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