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: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

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.