Similar Datasets
Project description:Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.
| S-EPMC7776864 | biostudies-literature
Project description:Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present HyPR-seq, a method to sensitively quantify the expression of 10 to 100 chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects non-polyadenylated and low-abundance transcripts, and can profile up to 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell type frequencies in tissue using low-abundance marker genes. By sensitively quantifying individual transcripts and directing sequencing power to genes of interest, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome scRNA-seq approaches for these applications. HyPR-seq will be a powerful method for targeted RNA profiling in single cells.
2020-09-16 | GSE158003 | GEO
Project description:Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present HyPR-seq, a method to sensitively quantify the expression of 10 to 100 chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects non-polyadenylated and low-abundance transcripts, and can profile up to 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell type frequencies in tissue using low-abundance marker genes. By sensitively quantifying individual transcripts and directing sequencing power to genes of interest, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome scRNA-seq approaches for these applications. HyPR-seq will be a powerful method for targeted RNA profiling in single cells.
2020-09-16 | GSE158002 | GEO
Project description:Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present HyPR-seq, a method to sensitively quantify the expression of 10 to 100 chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects non-polyadenylated and low-abundance transcripts, and can profile up to 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell type frequencies in tissue using low-abundance marker genes. By sensitively quantifying individual transcripts and directing sequencing power to genes of interest, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome scRNA-seq approaches for these applications. HyPR-seq will be a powerful method for targeted RNA profiling in single cells.
2020-09-16 | GSE158001 | GEO
Project description:HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes
| PRJNA663608 | ENA
Project description:HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes (HyPR-seq dataset)
| PRJNA663612 | ENA
Project description:HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes (scRNA-seq dataset)
| PRJNA663611 | ENA
Project description:HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes (bulk RNA-seq dataset)
| PRJNA663610 | ENA
Project description:Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays.
| S-EPMC4099319 | biostudies-literature
Project description:Chromosomal rearrangements are frequently monitored by fluorescence in situ hybridization (FISH) using large, recombinant DNA probes consisting of contiguous genomic intervals that are often distant from disease loci. We developed smaller, targeted, single-copy probes directly from the human genome sequence. These single-copy FISH (scFISH) probes were designed by computational sequence analysis of approximately 100-kb genomic sequences. ScFISH probes are produced by long PCR, then purified, labeled, and hybridized individually or in combination to human chromosomes. Preannealing or blocking with unlabeled, repetitive DNA is unnecessary, as scFISH probes lack repetitive DNA sequences. The hybridization results are analogous to conventional FISH, except that shorter probes can be readily visualized. Combinations of probes from the same region gave single hybridization signals on metaphase chromosomes. ScFISH probes are produced directly from genomic DNA, and thus more quickly than by recombinant DNA techniques. We developed single-copy probes for three chromosomal regions-the CDC2L1 (chromosome 1p36), MAGEL2 (chromosome 15q11.2), and HIRA (chromosome 22q11.2) genes-and show their utility for FISH. The smallest probe tested was 2290 bp in length. To assess the potential utility of scFISH for high-resolution analysis, we determined chromosomal distributions of such probes. Single-copy intervals of this length or greater are separated by an average of 29.2 and 22.3 kb on chromosomes 21 and 22, respectively. This indicates that abnormalities seen on metaphase chromosomes could be characterized with scFISH probes at a resolution greater than previously possible.
| S-EPMC311125 | biostudies-literature