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

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

PROVIDER: PRJNA700076 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13645917_1.fastq.gz	Fastqsanger.gz
	SRR13645917_2.fastq.gz	Fastqsanger.gz
	SRR13645918_1.fastq.gz	Fastqsanger.gz
	SRR13645918_2.fastq.gz	Fastqsanger.gz
	SRR13645919_1.fastq.gz	Fastqsanger.gz

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

Project description: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.

2021-02-07 | GSE166293 | GEO

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

Project description: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.

| S-EPMC8191787 | biostudies-literature

Multiplexed Single Cell gene expression profile of barcoded SUM159PT cells

Project description:Cancer is a heterogeneous disease, where multiple, phenotypically distinct subpopulations co-exist. Tumour evolution is a result of a complex interplay of genetic and epigenetic factors. To predict the molecular drivers of distinct cancer responses, we apply single-cell lineage tracing (scRNA-Seq of barcoded cells) on a triple-negative breast cancer model. SUM159PT cells infected with a lentiviral barcode library (Perturb-seq Library) were sorted according to the presence of BFP signal, treated or not with paclitaxel (PTX), multiplexed with MULTI-Seq protocol, and then processed by scRNA-Seq.

2024-05-24 | E-MTAB-13066 | biostudies-arrayexpress

Highly multiplexed and quantitative cell surface protein profiling using genetically barcoded antibodies

Project description:Next generation sequencing (NGS) allows for sensitive quantification of DNA and RNA. It would be highly desirable to have a systematic equivalent for assaying cellular protein levels on living cells. We present a highly multiplexed, quantitative, and inexpensive sequencing-based proteomic method using genetically barcoded antibodies called Phage-antibody Next Generation Sequencing (PhaNGS). We demonstrate the utility of PhaNGS by showing how a set of 144 targeted Fab-phage can reliably detect changes in 44 targeted cell surface proteins in drug sensitive and resistant B-cells, or upon induction of the Myc oncogene.

2018-02-12 | GSE102712 | GEO

Multiplexed Single Cell gene expression profile of barcoded SUM159PT cells

Project description:Multiplexed Single Cell gene expression profile of barcoded SUM159PT cells

| PRJEB63146 | ENA

RNA-Seq of multiplexed metastasis xenogfrates of barcoded cell line pools

Project description:RNA-Seq of multiplexed metastasis xenogfrates of barcoded cell line pools

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Project description:New method to measure tumor growth with programmable multiplexed CRISPR/Cas9-induced genotypes via ultra-deep sequencing of DNA barcoded tumors

2017-05-10 | GSE98207 | GEO

Multiplexed protein quantification with barcoded hydrogel microparticles.

Project description:We demonstrate the use of graphically encoded hydrogel microparticles for the sensitive and high-throughput multiplexed detection of clinically relevant protein panels in complex media. Combining established antibody capture techniques with advances in both microfluidic synthesis and analysis, we detected 1-8 pg/mL amounts of three cytokines (interleuken-2, interleuken-4, and tumor necrosis factor alpha) in single and multiplexed assays without the need for filtration or blocking agents. A range of hydrogel porosities was investigated to ensure rapid diffusion of targets and reagents into the particle as well as to maintain the structural integrity of particles during rinsing procedures and high-velocity microfluidic scanning. Covalent incorporation of capture antibodies using a heterobifunctional poly(ethylene glycol) linker enabled one-step synthesis and functionalization of particles using only small amounts of valuable reagents. In addition to the use of three separate types of single-probe particles, the flexibility of the stop-flow lithography (SFL) method was leveraged to spatially segregate the three probes for the aforementioned target set on an individual encoded particle, thereby demonstrating the feasibility of single-particle diagnostic panels. This study establishes the gel-particle platform as a versatile tool for the efficient quantification of protein targets and significantly advances efforts to extend the advantages of both hydrogel substrates and particle-based arrays to the field of clinical proteomics.

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Magnetic barcoded hydrogel microparticles for multiplexed detection.

Project description:Magnetic polymer particles have been used in a wide variety of applications ranging from targeting and separation to diagnostics and imaging. Current synthesis methods have limited these particles to spherical or deformations of spherical morphologies. In this paper, we report the use of stop flow lithography to produce magnetic hydrogel microparticles with a graphical code region, a probe region, and a magnetic tail region. These anisotropic multifunctional magnetic polymer particles are an enhanced version of previously synthesized "barcoded" particles (Science, 2007, 315, 1393-1396) developed for the sensitive and rapid multiplexed sensing of nucleic acids. The newly added magnetic region has acquired dipole moments in the presence of weak homogeneous magnetic fields, allowing the particles to align along the applied field direction. The novel magnetic properties have led to practical applications in the efficient orientation and separation of the barcoded microparticles during biological assays without disrupting detection capabilities.

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Project description:We developed an amplified single-molecule RNA FISH method called clampFISH 2.0. To validate its performance, we used clampFISH 2.0 on two cell lines, WM989 A6-G3 cells and WM989 A6-G3 cells grown in vemurafenib, and across 10 genes, compared clampFISH 2.0 spot counts to the FPKM values from bulk RNA sequencing.

2022-08-23 | GSE211491 | GEO

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