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

Eswar P. R. Iyer, Sukanya Punthambaker, Songlei Liu, Kunal Jindal, Michael Farrell, Jernej Murn, Thomas Ferrante, Stewart Rudnicki, Richie E. Kohman, Asmamaw T. Wassie, Daniel Goodwin, Fei Chen, Shahar Alon, Anubhav Sinha, Denitsa Milanova, Liviu Aron, Conor Camplisson, Alex Skrynnyk, Paul Louis Reginato, Nick Conway, John Aach, Bruce Yankner, Edward S. Boyden, George M. Church
doi: https://doi.org/10.1101/281121
Eswar P. R. Iyer
1Department of Genetics, Harvard Medical School, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Sukanya Punthambaker
1Department of Genetics, Harvard Medical School, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Songlei Liu
1Department of Genetics, Harvard Medical School, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Kunal Jindal
1Department of Genetics, Harvard Medical School, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Michael Farrell
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Jernej Murn
3University of California, Riverside, Riverside, CA, USA
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Thomas Ferrante
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Stewart Rudnicki
1Department of Genetics, Harvard Medical School, Boston, MA, USA
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Richie E. Kohman
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Asmamaw T. Wassie
5Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
6McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA, USA
7Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA
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Daniel Goodwin
5Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
6McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA, USA
7Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA
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Fei Chen
9Broad Institute, Massachusetts Institute of Technology, Cambridge, MA, USA
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Shahar Alon
4MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, USA
6McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA, USA
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Anubhav Sinha
8Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA
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Denitsa Milanova
1Department of Genetics, Harvard Medical School, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Liviu Aron
1Department of Genetics, Harvard Medical School, Boston, MA, USA
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Conor Camplisson
1Department of Genetics, Harvard Medical School, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Alex Skrynnyk
1Department of Genetics, Harvard Medical School, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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Paul Louis Reginato
1Department of Genetics, Harvard Medical School, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
5Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
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Nick Conway
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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John Aach
1Department of Genetics, Harvard Medical School, Boston, MA, USA
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Bruce Yankner
1Department of Genetics, Harvard Medical School, Boston, MA, USA
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Edward S. Boyden
4MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, USA
5Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
6McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA, USA
7Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA
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George M. Church
1Department of Genetics, Harvard Medical School, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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  • For correspondence: gchurch@genetics.med.harvard.edu
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Abstract

We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallel In-Situ analysis (BOLORAMIS), a reverse-transcription (RT)-free method for spatially-resolved, targeted, in-situ RNA identification of single or multiple targets. For this proof of concept, we have profiled 154 distinct coding and small non-coding transcripts ranging in sizes 18 nucleotide (nt) in length and upwards, from over 200, 000 individual human induce d pluripotent stem cells (iPSC) and demonstrated compatibility with multiplexed detection, enabled by fluorescent in-situ sequencing. We use BOLORAMIS data to identify differences in spatial localization and cell-to-cell expression heterogeneity. Our results demonstrate BOLORAMIS to be a generalizable toolset for targeted, in-situ detection of coding and small non-coding RNA for single or multiplexed applications.

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Posted March 13, 2018.
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Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses
Eswar P. R. Iyer, Sukanya Punthambaker, Songlei Liu, Kunal Jindal, Michael Farrell, Jernej Murn, Thomas Ferrante, Stewart Rudnicki, Richie E. Kohman, Asmamaw T. Wassie, Daniel Goodwin, Fei Chen, Shahar Alon, Anubhav Sinha, Denitsa Milanova, Liviu Aron, Conor Camplisson, Alex Skrynnyk, Paul Louis Reginato, Nick Conway, John Aach, Bruce Yankner, Edward S. Boyden, George M. Church
bioRxiv 281121; doi: https://doi.org/10.1101/281121
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Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses
Eswar P. R. Iyer, Sukanya Punthambaker, Songlei Liu, Kunal Jindal, Michael Farrell, Jernej Murn, Thomas Ferrante, Stewart Rudnicki, Richie E. Kohman, Asmamaw T. Wassie, Daniel Goodwin, Fei Chen, Shahar Alon, Anubhav Sinha, Denitsa Milanova, Liviu Aron, Conor Camplisson, Alex Skrynnyk, Paul Louis Reginato, Nick Conway, John Aach, Bruce Yankner, Edward S. Boyden, George M. Church
bioRxiv 281121; doi: https://doi.org/10.1101/281121

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