Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses
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gkab120.pdf
Description
Published version
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4.17 MB
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Checksum (MD5)
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Author(s)
Liu, Songlei
Punthambaker, Sukanya
Iyer, Eswar PR
Ferrante, Thomas
Goodwin, Daniel
Fürth, Daniel
Pawlowski, Andrew C
Jindal, Kunal
Tam, Jenny M
Mifflin, Lauren
Date Issued
2021
Journal
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Citation
Liu, Songlei, Punthambaker, Sukanya, Iyer, Eswar PR, Ferrante, Thomas, Goodwin, Daniel et al. 2021. "Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses." Nucleic Acids Research, 49 (10).
Version
Final published version
Abstract
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.
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.
MIT Department
McGovern Institute for Brain Research at MIT
Program in Media Arts and Sciences (Massachusetts Institute of Technology)
Harvard University--MIT Division of Health Sciences and Technology
Massachusetts Institute of Technology. Department of Biological Engineering
Koch Institute for Integrative Cancer Research at MIT
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
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Creative Commons Attribution 4.0 International license
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DOI of Published Version
https://doi.org/10.1093/NAR/GKAB120
