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

We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallel In Situ 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 POL2RA respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglia 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. Paired end RNA sequencing of HMC3 cells, PGP1 fibroblasts, and MCF7 cells. Sequencing was performed on Illumina platforms. Reads were aligned to GRCh38 GENCODE release 27. Counts for PGP1 fibroblasts and MCF7 cells were used in the probe number test experiments. Counts for HMC3 were used in the multiplexed RNA detection experiments.