Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses. Issue 10 (8th March 2021)

• Record Type: Journal Article
• Authors: Liu, Songlei; Punthambaker, Sukanya; Iyer, Eswar P R; 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
• Is Part Of: Nucleic acids research. Volume 49:Issue 10(2021)
• Page Start: e58
• Page End: e58
• Publication Date: 2021-03-08
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkab120
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: We present b arcoded o ligonucleotides l igated o n R NA a mplified for m ultiplexed and parallel i n s itu 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.