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

Liu, Songlei, Punthambaker, Sukanya, Iyer, Eswar PR, Ferrante, Thomas, Goodwin, Daniel, Fürth, Daniel, Pawlowski, Andrew C, Jindal, Kunal, Tam, Jenny M, Mifflin, Lauren, Alon, Shahar, Sinha, Anubhav, Wassie, Asmamaw T, Chen, Fei, Cheng, Anne, Willocq, Valerie, Meyer, Katharina, Ling, King-Hwa, Camplisson, Conor K, Kohman, Richie E, Aach, John, Lee, Je Hyuk, Yankner, Bruce A, Boyden, Edward S, Church, George M (March 2021) Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses. Nucleic Acids Research. ISSN 0305-1048

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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.

Item Type:	Paper
Subjects:	bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA expression bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > oligonucleotide organs, tissues, organelles, cell types and functions > cell types and functions > cell types > stem cells organs, tissues, organelles, cell types and functions > cell types and functions > cell types > stem cells organs, tissues, organelles, cell types and functions > cell types and functions > cell types > stem cells
CSHL Authors:	Lee, Je Hyuk Furth, Daniel
Communities:	CSHL labs > Lee lab
SWORD Depositor:	CSHL Elements
Depositing User:	CSHL Elements
Date:	8 March 2021
Date Deposited:	23 Apr 2021 17:28
Last Modified:	04 Aug 2021 15:55
PMCID:	PMC8191787
URI:	https://repository.cshl.edu/id/eprint/39919

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