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RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

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Published on: August 7, 2021

A Detailed Protocol for Subcellular RNA Sequencing (subRNA-seq).

Andreas Mayer¹, L Stirling Churchman²

¹Max Planck Institute for Molecular Genetics, Berlin, Germany.

Current Protocols in Molecular Biology

|October 3, 2017

Summary

This summary is machine-generated.

Subcellular RNA sequencing (subRNA-seq) quantifies RNA in different cell parts. This method overcomes limitations of standard RNA sequencing, revealing nuclear and cytoplasmic RNA profiles.

Keywords:

Cell fractionation RNA polymerase II (Pol II)RNA processing nascent RNA next-generation sequencing subcellular RNA-seq transcription

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Area of Science:

Molecular Biology
Genomics
Cell Biology

Background:

RNA molecules are distributed across cellular compartments at various maturation stages.
Standard RNA sequencing (RNA-seq) primarily detects abundant, stable cytoplasmic RNAs, overlooking nuclear and nascent transcripts.
A method is needed to comprehensively analyze RNA distribution and processing within cells.

Purpose of the Study:

To present a detailed protocol for subcellular RNA sequencing (subRNA-seq).
To enable quantitative measurement of RNA polymerase II-generated RNAs across cellular compartments (chromatin, nucleoplasm, cytoplasm).
To provide insights into RNA processing, maturation, and subcellular localization.

Main Methods:

Subcellular fractionation of mammalian cells before RNA isolation.
Preparation of sequencing libraries from fractionated cellular components.
High-throughput sequencing to analyze RNA identity, abundance, and distribution.

Main Results:

Subcellular RNA sequencing (subRNA-seq) effectively quantifies RNAs from chromatin, nucleoplasm, and cytoplasm.
The protocol allows for the study of nascent RNAs through deep sequencing of chromatin-associated RNAs.
Libraries for subRNA-seq can be generated within 5 days.

Conclusions:

SubRNA-seq offers a powerful approach to comprehensively study the transcriptome within specific cellular compartments.
This method enhances our understanding of RNA metabolism, processing, and localization in eukaryotic cells.
SubRNA-seq provides a valuable tool for investigating nuclear and cytoplasmic RNA dynamics.