Our Approaches

The Bose lab is a mechanistic biology lab interested in how gene expression can be regulated by enhancer RNA. We use diverse techniques to study how RNA interacts with these epigenetic machines over a range of scales, from functional genomics at a cellular level, to biochemistry and cryo-electron microscopy to understand molecular-mechanisms and structural architectures.

 

Functional Genomics

We use a variety of modern functional genomics approaches in the lab, underpinned by next-generation sequencing (seq) and analysis. These include:

  • PAR-CLIP/CRAC and Illumina sequencing to identify RNAs interacting with epigenetic-enzymes.

  • ChIPseq to study sites of chromatin binding across the genome and

  • Nascent RNA sequencing to identify sites of non-coding and eRNA transcription.

  • SHAPE-MaP to map RNA secondary structure.

We also apply a variety of CRISPR/Cas based approaches to develop model systems in cells. In combination, these approaches provide a powerful way to characterise and test the interactions made by our protein and RNA targets across the genome.

Our facilities include:

  • Nanopore MinION for direct RNA sequencing

  • UV-X-linker, Qubit, qPCR thermalcycler and Diagenode Bioruptor.

We also work with the University of Sheffield Genomics Lab and the Bioinformatics Core

Luc sequencing nascent RNA using the Nanopore MinION

Luc sequencing nascent RNA using the Nanopore MinION

We’re pretty excited about Cryo-EM

We’re pretty excited about Cryo-EM

Cryo-EM

We use cryo-EM to analyse the structural organisation of epigenetic enzyme complexes, shedding light on their interactions with  non-coding RNAs and engagement with chromatin. The approach lets us study the molecular details of interactions between eRNAs and the enzymes they bind to in unprecedented detail.

We use the University of Sheffield on-site cryo-EM facility. The facility comprises:

  • A 200kV Tecnai Arctica electron microscope equipped with Falcon 3 direct electron detector and phase plate,

  • Two 100kV electron microscopes

  • Dedicated GPU and CPU image processing clusters.

We also have two full-time, experienced facility managers for support and user training, and regular cryo-EM group meetings provide support and feedback, as well as a stimulating forum to discuss the latest technological advances. For high-resolution data collection, we have regular block allocation group (BAG) access to the eBIC Krios facility at Diamond.

Biochemistry

All of the work in the lab is underpinned by modern biochemistry, molecular biology and cell biology. We carry out extensive biochemical characterisations of the interactions between eRNAs and epigenetic enzymes, both in vitro and in cells. We also characterise the effect of eRNA binding on the activities of these enzymes using enzymatic assays. Our work often requires the expression, purification and analysis of the large multi-component complexes that we study.

We are well equipped for all forms of biochemistry, including:

  • AKTA Pure 25M FPLC with multi wavelength detector

  • AKTA Start FPLC

  • Protein expression in insect cells using Baculovirus and mammalian expression systems.

  • Bacterial protein expression.

  • Hidex Sense Beta Plate reader

    • We made a video for LabLogic talking about the plate reader - you can watch it here :)

  • GE Typhoon fluorescence/Phosphorimager

  • Dedicated radiation facility.

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Cell Biology

We have a Brand new tissue culture facility for all our cell biology work. We maintain a variety of cell lines, from Jurkat T-ALL cells to model neuronal cell lines and stem cell. This lets us build models to study diverse aspects of enhancer and eRNA activity.

Ouf facility includes:

  • 2x MSC advantage BSL2 laminar flow hoods

  • ThermoFisher Neon Electroporator

  • 2 HERAsafe 150i Copper lined CO2 incubators