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Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation.

ABSTRACT: Whereas techniques to map chromatin-bound proteins are well developed, mapping chromatin-associated RNAs remains a challenge. Here, we describe Reverse Transcribe and Tagment (RT&Tag), in which RNAs associated with a chromatin epitope are targeted by an antibody followed by a protein A-Tn5 transposome. Localized reverse transcription generates RNA/cDNA hybrids that are subsequently tagmented by Tn5 transposases for downstream sequencing. We demonstrate the utility of RT&Tag in Drosophila cells for capturing the noncoding RNA roX2 with the dosage compensation complex and maturing transcripts associated with silencing histone modifications. We also show that RT&Tag can detect N6-methyladenosine-modified mRNAs, and show that genes producing methylated transcripts are characterized by extensive promoter pausing of RNA polymerase II. The high efficiency of in situ antibody tethering and tagmentation makes RT&Tag especially suitable for rapid low-cost profiling of chromatin-associated RNAs.

SUBMITTER: Khyzha N

PROVIDER: S-EPMC9636022 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation.

Khyzha Nadiya N Henikoff Steven S Ahmad Kami K

Nature methods 20221003 11

Whereas techniques to map chromatin-bound proteins are well developed, mapping chromatin-associated RNAs remains a challenge. Here, we describe Reverse Transcribe and Tagment (RT&Tag), in which RNAs associated with a chromatin epitope are targeted by an antibody followed by a protein A-Tn5 transposome. Localized reverse transcription generates RNA/cDNA hybrids that are subsequently tagmented by Tn5 transposases for downstream sequencing. We demonstrate the utility of RT&Tag in Drosophila ...[more]

PMID: 36192462

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Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation.

Project description:Whereas techniques to map chromatin-bound proteins are well-developed, mapping chromatin-associated RNAs remains a challenge. Here we describe Reverse Transcribe & Tagment (RT&Tag), in which RNAs associated with a chromatin epitope are targeted by an antibody followed by a protein A-Tn5 transposome. Localized reverse transcription generates and the Tn5 transposase tagments RNA/cDNA hybrids. We demonstrate the utility of RT&Tag in Drosophila cells for capturing the noncoding RNA roX2 with the dosage compensation complex and maturing transcripts associated with silencing histone modifications. We also show that RT&Tag can detect N6-methyladenosine (m6A)-modified mRNAs, and show that genes producing methylated transcripts are characterized by extensive promoter pausing of RNA polymerase II. The high efficiency of in situ antibody tethering and tagmentation makes RT&Tag especially suitable for rapid low-cost profiling chromatin-associated RNAs from small samples.

2022-07-18 | GSE195654 | GEO

Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation.

Project description:Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation.

| PRJNA801630 | ENA

Simplified Epigenome Profiling Using Antibody-tethered Tagmentation.

Project description:We previously introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling method in which antibody tethering of the Tn5 transposase to a chromatin epitope of interest maps specific chromatin features in small samples and single cells. With CUT&Tag, intact cells or nuclei are permeabilized, followed by successive addition of a primary antibody, a secondary antibody, and a chimeric Protein A-Transposase fusion protein that binds to the antibody. Addition of Mg++ activates the transposase and inserts sequencing adapters into adjacent DNA in situ. We have since adapted CUT&Tag to also map chromatin accessibility by simply modifying the transposase activation conditions when using histone H3K4me2, H3K4me3, or Serine-5-phosphorylated RNA Polymerase II antibodies. Using these antibodies, we redirect the tagmentation of accessible DNA sites to produce chromatin accessibility maps with exceptionally high signal-to-noise and resolution. All steps from nuclei to amplified sequencing-ready libraries are performed in single PCR tubes using non-toxic reagents and inexpensive equipment, making our simplified strategy for simultaneous chromatin profiling and accessibility mapping suitable for the lab, home workbench, or classroom.

| S-EPMC8250384 | biostudies-literature

Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation.

Project description:Chromatin accessibility mapping is a powerful approach to identify potential regulatory elements. A popular example is ATAC-seq, whereby Tn5 transposase inserts sequencing adapters into accessible DNA ('tagmentation'). CUT&Tag is a tagmentation-based epigenomic profiling method in which antibody tethering of Tn5 to a chromatin epitope of interest profiles specific chromatin features in small samples and single cells. Here, we show that by simply modifying the tagmentation conditions for histone H3K4me2 or H3K4me3 CUT&Tag, antibody-tethered tagmentation of accessible DNA sites is redirected to produce chromatin accessibility maps that are indistinguishable from the best ATAC-seq maps. Thus, chromatin accessibility maps can be produced in parallel with CUT&Tag maps of other epitopes with all steps from nuclei to amplified sequencing-ready libraries performed in single PCR tubes in the laboratory or on a home workbench. As H3K4 methylation is produced by transcription at promoters and enhancers, our method identifies transcription-coupled accessible regulatory sites.

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Direct RNA targeted in situ sequencing for transcriptomic profiling in tissue.

Project description:Highly multiplexed spatial mapping of transcripts within tissues allows for investigation of the transcriptomic and cellular diversity of mammalian organs previously unseen. Here we explore a direct RNA (dRNA) detection approach incorporating the use of padlock probes and rolling circle amplification in combination with hybridization-based in situ sequencing chemistry. We benchmark a High Sensitivity Library Preparation Kit from CARTANA that circumvents the reverse transcription needed for cDNA-based in situ sequencing (ISS) via direct RNA detection. We found a fivefold increase in transcript detection efficiency when compared to cDNA-based ISS and also validated its multiplexing capability by targeting a curated panel of 50 genes from previous publications on mouse brain sections, leading to additional data interpretation such as de novo cell clustering. With this increased efficiency, we also found to maintain specificity, multiplexing capabilities and ease of implementation. Overall, the dRNA chemistry shows significant improvements in target detection efficiency, closing the gap to other fluorescent in situ hybridization-based technologies and opens up possibilities to explore new biological questions previously not possible with cDNA-based ISS.

| S-EPMC9106737 | biostudies-literature

Author Correction: Direct RNA targeted in situ sequencing for transcriptomic profiling in tissue.

Project description: Not available

| S-EPMC10850151 | biostudies-literature

Mapping histone modifications in low cell number and single cells using antibody-guided chromatin tagmentation (ACT-seq).

Project description:Modern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. Here we describe ACT-seq, a streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present an attractive alternative to existing techniques for mapping epigenetic marks in single cells.

| S-EPMC6702168 | biostudies-literature

RNA sequencing by direct tagmentation of RNA/DNA hybrids.

Project description:Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

| S-EPMC7022195 | biostudies-literature

Molecular Profiling of Druggable Targets in Clear Cell Renal Cell Carcinoma Through Targeted RNA Sequencing.

Project description:Clear cell renal cell carcinoma (ccRCC) comprises more than 80% of all renal cancers and when metastasized leads to a 5-year survival rate of only 10%. The high rate of therapy failure and resistance development calls for reliable methods that provide information on the actionable biological pathways and predict optimal treatment protocols for individual patients. We here applied targeted RNA sequencing (t/RNA-NGS) using single molecule Molecular Inversion Probes on tumor nephrectomy samples of five ccRCC patients, comparing tumor with healthy kidney tissues. Transcriptome profiling focused on expression of genes with involvement in ccRCC biology that can be targeted with clinically available drugs. Results confirm high expression of vascular endothelial growth factor-A (VEGF-A) in tumor tissue relative to healthy-appearing kidney, in line with the angiogenic nature of ccRCC. PDGFRα and KIT, targets of the multi-kinase inhibitor sunitinib which is one of the current choices of first-line drug in metastasized ccRCC patients, were expressed at relatively low levels in tumor tissues, whereas significantly increased in normal kidney. Of all measured druggable tyrosine kinases, MET, AXL, or EGFR were expressed at higher levels in tumors than in normal kidney tissues, although intertumor differences were observed. Using cancer cell lines we show that t/RNA-NGS gene expression profiles can be used to predict in vitro sensitivity to targeted drugs. In conclusion, t/RNA-NGS analysis may provide insights into the (druggable) molecular make-up of individual renal cancers, and may guide personalized therapy of renal cell cancers.

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Transposase-assisted tagmentation of RNA/DNA hybrid duplexes.

Project description:Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase-assisted RNA/DNA hybrids Co-tagmEntation (termed 'TRACE-seq') are compared to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis. At the meantime, TRACE-seq enables a cost-effective one-tube library construction protocol and hence is more rapid (within 6 hr) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.

| S-EPMC7402673 | biostudies-literature

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