Butt-seq: a new method for facile profiling of transcription
Albert D Yu1, Michael Rosbash2
1Department of Biology, Howard Hughes Medical Institute, National Center for Behavioral Genomics, Brandeis University, Waltham, Massachusetts 02453, USA.
Genes & Development
|May 10, 2023
Summary
We developed Butt-seq, a rapid and low-input sequencing method for analyzing nascent RNA transcription. This technique improves upon existing methods, enabling high-resolution transcriptional studies with minimal cellular material.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Existing sequencing methods for nascent RNA transcription often require substantial cell input and lengthy protocols.
- Resolving RNA polymerase position genome-wide is crucial for understanding transcriptional regulation.
Purpose of the Study:
- To develop a more efficient and sensitive sequencing technique for analyzing nascent RNA transcription.
- To overcome the limitations of high input material requirements and lengthy protocols in current methods.
Main Methods:
- Leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT).
- Developed Butt-seq (bulk analysis of nascent transcript termini sequencing) for rapid library preparation.
- Demonstrated Butt-seq's ability to produce libraries from purified nascent RNA in 6 hours and from as few as 10,000 cells.
Main Results:
- Butt-seq offers at least a 10-fold improvement over existing techniques in terms of speed and input material.
- Showed that superelongation complex (SEC) inhibition leads to upstream shifts in promoter-proximal pausing, correlated with subnucleosomal fragments.
- Applied Butt-seq to study circadian regulation of transcription in Drosophila melanogaster.
Conclusions:
- Butt-seq is a simple, powerful, and high-resolution technique for analyzing nascent RNA transcription.
- The method significantly reduces time and cell input, making transcription studies more accessible.
- Butt-seq provides novel insights into transcriptional regulation, including promoter-proximal pausing and circadian rhythms.
Related Concept Videos
02:24Ribosome Profiling
3.6K
Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
3.6K
03:21RNA-seq
10.1K
RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
10.1K