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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.

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