Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses

Songlei Liu, Sukanya Punthambaker, Eswar P.R. Iyer, Thomas Ferrante, Daniel Goodwin, Daniel Fürth, Andrew C. Pawlowski, Kunal Jindal, Jenny M. Tam, Lauren Mifflin, Shahar Alon, Anubhav Sinha, Asmamaw T. Wassie, Fei Chen, Anne Cheng, Valerie Willocq, Katharina Meyer, King Hwa Ling, Conor K. Camplisson, Richie E. KohmanJohn Aach, Je Hyuk Lee, Bruce A. Yankner, Edward S. Boyden, George M. Church

Research output: Contribution to journalArticlepeer-review

35 Scopus citations


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.

Original languageEnglish
Article numbere58
JournalNucleic Acids Research
Issue number10
StatePublished - 4 Jun 2021
Externally publishedYes

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Publisher Copyright:
© 2021 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research.


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