Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

MERR APEX-seq protocol for profiling the subcellular nascent transcriptome in mammalian cells.

ABSTRACT: Knowledge about the spatial organization of RNAs in eukaryotic cells is crucial for understanding their functions. Here, we present a detailed MERR APEX-seq protocol to achieve spatiotemporally resolved mapping of the subcellular transcriptome in cultured mammalian cells. This protocol provides detailed description of constructing cell lines stably expressing APEX2, immunofluorescence characterization, MERR APEX labeling, enrichment of biotinylated RNA, library construction and high-throughput sequencing, and MERR APEX-seq data analysis. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).¹.

SUBMITTER: Li R

PROVIDER: S-EPMC9898802 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

MERR APEX-seq protocol for profiling the subcellular nascent transcriptome in mammalian cells.

Li Ran R Zou Peng P

STAR protocols 20230124 1

Knowledge about the spatial organization of RNAs in eukaryotic cells is crucial for understanding their functions. Here, we present a detailed MERR APEX-seq protocol to achieve spatiotemporally resolved mapping of the subcellular transcriptome in cultured mammalian cells. This protocol provides detailed description of constructing cell lines stably expressing APEX2, immunofluorescence characterization, MERR APEX labeling, enrichment of biotinylated RNA, library construction and high-throughput s ...[more]

PMID: 36853684

Similar Datasets

Atlas of Subcellular RNA Localization Revealed by APEX-Seq.

Project description:We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

| S-EPMC6786773 | biostudies-literature

Protocol for affordable and efficient profiling of nascent RNAs in bread wheat using GRO-seq.

Project description:Exorbitant sequencing cost is one of the main obstacles limiting the widespread application of Global Run-On sequencing (GRO-seq) to detect transcriptional activity. Here, we describe a more efficient and affordable protocol for GRO-seq that incorporates an rRNA removal step after nuclear RNA isolation and before nascent RNA immunoprecipitation. We have successfully applied this protocol to profile enhancer transcription in allohexaploid bread wheat and increased the proportion of valid data by 20 times. For complete details on the use and execution of this protocol, please refer to Xie et al. (2022).

| S-EPMC9463444 | biostudies-literature

Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing.

Project description:Transcription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5'splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.

| S-EPMC4410947 | biostudies-literature

Protocol for Proximity-Dependent Proteomic Profiling in Yeast Cells by APEX and Alk-Ph Probe.

Project description:Alk-Ph is a clickable APEX2 substrate developed for spatially restricted protein/RNA labeling in intact yeast cells. Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. Using the yeast mitochondria as an example, we provide guidelines to achieve high-resolution mapping of subcellular yeast proteome and transcriptome. For complete details on the use and execution of this protocol, please refer to Li et al. (2020).

| S-EPMC7757286 | biostudies-literature

Transcriptome-wide organization of subcellular microenvironments revealed by ATLAS-Seq.

Project description:Subcellular organization of RNAs and proteins is critical for cell function, but we still lack global maps and conceptual frameworks for how these molecules are localized in cells and tissues. Here, we introduce ATLAS-Seq, which generates transcriptomes and proteomes from detergent-free tissue lysates fractionated across a sucrose gradient. Proteomic analysis of fractions confirmed separation of subcellular compartments. Unexpectedly, RNAs tended to co-sediment with other RNAs in similar protein complexes, cellular compartments, or with similar biological functions. With the exception of those encoding secreted proteins, most RNAs sedimented differently than their encoded protein counterparts. To identify RNA binding proteins potentially driving these patterns, we correlated their sedimentation profiles to all RNAs, confirming known interactions and predicting new associations. Hundreds of alternative RNA isoforms exhibited distinct sedimentation patterns across the gradient, despite sharing most of their coding sequence. These observations suggest that transcriptomes can be organized into networks of co-segregating mRNAs encoding functionally related proteins and provide insights into the establishment and maintenance of subcellular organization.

| S-EPMC7293051 | biostudies-literature

Metabolic incorporation of electron-rich ribonucleosides enhances APEX-seq for profiling spatially restricted nascent transcriptome

Project description:We present MERR APEX-seq, a method for newly transcribed RNAs subcellular profiling combined metabolic incorporation of electron-rich ribonucleosides, 6-thioguanosine and 4-thiouridine, with the peroxidase-mediated RNA labeling method, APEX-seq. MERR APEX-seq offers both high spatial specificity and high coverage in the mitochondrial matrix and at the endoplasmic reticulum membrane. Application of MERR APEX-seq at nuclear lamina of human cells reveals that the mRNA components tend to encode for transcripts processing related proteins. MERR APEX-seq with high spatial specificity and high coverage could be widely used to expand our knowledge of RNA localization and function at subcellular compartments.

2022-01-03 | GSE192739 | GEO

Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide.

Project description:The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide-resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.

| S-EPMC5152764 | biostudies-literature

Atlas of Subcellular RNA Localization Revealed by APEX-seq

2019-06-20 | GSE116008 | GEO

Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells.

Project description:To demonstrate the benefits of RNA-Seq over microarray in transcriptome profiling, both RNA-Seq and microarray analyses were performed on RNA samples from a human T cell activation experiment. In contrast to other reports, our analyses focused on the difference, rather than similarity, between RNA-Seq and microarray technologies in transcriptome profiling. A comparison of data sets derived from RNA-Seq and Affymetrix platforms using the same set of samples showed a high correlation between gene expression profiles generated by the two platforms. However, it also demonstrated that RNA-Seq was superior in detecting low abundance transcripts, differentiating biologically critical isoforms, and allowing the identification of genetic variants. RNA-Seq also demonstrated a broader dynamic range than microarray, which allowed for the detection of more differentially expressed genes with higher fold-change. Analysis of the two datasets also showed the benefit derived from avoidance of technical issues inherent to microarray probe performance such as cross-hybridization, non-specific hybridization and limited detection range of individual probes. Because RNA-Seq does not rely on a pre-designed complement sequence detection probe, it is devoid of issues associated with probe redundancy and annotation, which simplified interpretation of the data. Despite the superior benefits of RNA-Seq, microarrays are still the more common choice of researchers when conducting transcriptional profiling experiments. This is likely because RNA-Seq sequencing technology is new to most researchers, more expensive than microarray, data storage is more challenging and analysis is more complex. We expect that once these barriers are overcome, the RNA-Seq platform will become the predominant tool for transcriptome analysis.

| S-EPMC3894192 | biostudies-literature

Efficient and strand-specific profiling of replicating chromatin with enrichment and sequencing of protein-associated nascent DNA in mammalian cells.

Project description:Faithful duplication of both genetic and epigenetic information is essential for all eukaryotic cells. DNA replication initiates from replication origins and proceeds bidirectionally but asymmetrically, with the leading strand being synthesized continuously and the lagging strand discontinuously as Okazaki fragments by distinct DNA polymerases. Unraveling the underlying mechanisms of chromatin replication at both strands is crucial to better understand DNA replication and its coupled processes, including nucleosome assembly, sister chromatid cohesion and DNA mismatch repair. Here we describe the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method to analyze the enrichment of proteins of interest, including histones and their modifications at replicating chromatin in a strand-specific manner in mammalian cells. Briefly, cells are pulsed with the thymidine analog bromodeoxyuridine to label newly synthesized DNA. After cell permeabilization, the target proteins are sequentially bound by antibodies and protein A-fused transposase, which digests and tags genomic DNA of interest once activated by magnesium. The strand specificity is preserved through oligo-replacement. Finally, the resulting double-strand DNA is denatured and immunoprecipitated with antibodies against bromodeoxyuridine to enrich nascent DNA associated with proteins of interest. After PCR amplification and next-generation sequencing, the mapped reads are used to calculate the relative enrichment of the target proteins around replication origins. Compared with alternative methods, the eSPAN protocol is simple, cost-effective and sensitive, even in a relatively small number of mammalian cells. The whole procedures from cell collection to generation of sequencing-ready libraries can be completed in 2 days.

| S-EPMC9261918 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data