PMID- 33693773 OWN - NLM STAT- MEDLINE VI - 49 IP - 10 TI - Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses. PG - e58 CI - © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. LA - eng PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PL - England TA - Nucleic Acids Res JT - Nucleic acids research JID - 0411011 IS - 1362-4962 (Electronic) LID - 10.1093/nar/gkab120 [doi] FAU - Liu, Songlei AU - Liu S AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. FAU - Punthambaker, Sukanya AU - Punthambaker S AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Iyer, Eswar P R AU - Iyer EPR AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Ferrante, Thomas AU - Ferrante T AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Goodwin, Daniel AU - Goodwin D AD - McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. FAU - Fürth, Daniel AU - Fürth D AD - Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. FAU - Pawlowski, Andrew C AU - Pawlowski AC AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Jindal, Kunal AU - Jindal K AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Tam, Jenny M AU - Tam JM AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Mifflin, Lauren AU - Mifflin L AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. FAU - Alon, Shahar AU - Alon S AD - McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. FAU - Sinha, Anubhav AU - Sinha A AD - McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Harvard-MIT Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. FAU - Wassie, Asmamaw T AU - Wassie AT AD - McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. FAU - Chen, Fei AU - Chen F AD - Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Broad Institute, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. FAU - Cheng, Anne AU - Cheng A AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Willocq, Valerie AU - Willocq V AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Meyer, Katharina AU - Meyer K AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. FAU - Ling, King-Hwa AU - Ling KH AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. AD - Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. FAU - Camplisson, Conor K AU - Camplisson CK AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Kohman, Richie E AU - Kohman RE AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. FAU - Aach, John AU - Aach J AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. FAU - Lee, Je Hyuk AU - Lee JH AD - Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. FAU - Yankner, Bruce A AU - Yankner BA AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. FAU - Boyden, Edward S AU - Boyden ES AD - McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. AD - Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. AD - Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USAHoward Hughes Medical Institute, Chevy Chase, MD 20815, USA. FAU - Church, George M AU - Church GM AD - Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. AD - Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. IS - 0305-1048 (Linking) RN - 0 (Oligonucleotides) RN - 63231-63-0 (RNA) SB - IM MH - Animals MH - Cell Line MH - Gene Expression Profiling/*methods MH - Humans MH - In Situ Hybridization, Fluorescence/*methods MH - Mice MH - Oligonucleotides/*chemistry MH - RNA/*analysis MH - Single-Cell Analysis/*methods PMC - PMC8191787 DCOM- 20210706 LR - 20210914 DP - 20210604 AB - 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.