2018 Institute Annual Research Report Facilities 442-53 Facilities

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Bioinformatics Biological Chemistry Biological Support Unit Flow Cytometry Gene Targeting

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Imaging Lipidomics Mass Spectrometry Sequencing

43 Facilities

Bioinformatics

Research increasingly includes the creation of large amounts of data and the use of computers to manage and process that information. The Bioinformatics facility provides Simon Andrews infrastructure to support the analysis Facility head of biological data. We provide guidance and training in data analysis, statistics Facility members and data management to both internal Biological statistician: and external groups. We also develop Anne Segonds-Pichon novel tools, and administer the Institute’s computing cluster. Bioinformaticians: Laura Biggins Capabilities Christel Krueger n An 800 node compute cluster with an Felix Krueger extensive collection of bioinformatics Steven Wingett software and pipelines. Training developer: n Jo Montgomery A range of custom software often focused on next generation sequencing, data visualisation and quality control.

n Experience in the processing, This year, we expanded our toolsets to management and analysis of large support technologies such as NMT-Seq biological data sets. and single-cell RNA-Seq, BS-Seq, and RNA– interaction data. n An extensive modular portfolio of bioinformatics training courses targeted at biologists.

Progress in 2018 Selected Impact Activities In 2018 the Bioinformatics group has n We established a new set of bioinformaticians from around greatly expanded its training programme. bioinformatics training bootcamps, and the surrounding We have established a set of larger training covering next generation sequencing, areas. bootcamps running over several days R programming and Linux systems n Two members of the group undertook that provide an in-depth introduction administration. to a number of relevant topics covering an extended trip to the Hunter n sequence analysis, programming and We organised the Second Cambridge Medical Research Institute, Australia, technical skills. As well as running these Bioinformatics Hackathon at the to run a series of bioinformatics on site at the Babraham Research Campus, Centre for Computing History, and statistics training courses and we have also run training courses for bringing together over 50 establish a longer term relationship academic and commercial groups in the between our institutes. UK, Germany, Spain and Australia.

Publications www.bioinformatics.babraham.ac.uk @babraham_bioinf n Clark, S.J. et al. (2018) scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells. Nat. Commun. 9(1):781 n Chovanec, P. et al. (2018). Unbiased quantification of immunoglobulin diversity at the DNA level with VDJ-seq. Nat. Protoc. 13(6):1232-1252 n Wingett, S.W. et al. (2018) FastQ Screen: A tool for multi-genome mapping and quality control. F1000Res. 7:1338

44 Biological Chemistry

The Biological Chemistry group provides investigating the chemical changes n Development of new reagents and support for scientists working at the which occur in connective tissues as analytical methods. interface between chemistry and we age. n Help and advice on any aspect of the biology. We bring an understanding of Capabilities application of chemistry/biochemistry to chemistry and its application to solving Jonathan Clark n Chemical synthesis of standards and the exploration of biological problems. biological problems along with the Facility head reagents which are not commercially capability to implement our suggestions Progress in 2018 available. using chemical and analytical tools. Facility members During 2018 we have supported groups n Analysis of biological molecules by mass throughout the Institute on a wide range In addition to our collaborations with Postdoctoral research spectroscopy. of varied projects. These have ranged the Institute’s research groups we are scientists: from synthetic chemistry projects to make Izabella Niewczas compounds which are not commercially Mel Stammers available through to developing new analytical methods to analyse lipids in cell extracts. In addition to these activities we have also continued to run routine lipid analysis for a number of groups, both within the Institute and externally. Separately in the connective tissue project, we have shown for the first time that the chemistry of collagen dynamically changes in response to stretching. In this work we have been able to describe the changes that occur to this chemistry with age and provide an explanation for the changes in the physical properties of tendons observed in ageing and in diabetes.

Selected Impact Activities n Through 2018 we have run many commercial PIP3 analysis samples for a number of pharmaceutical companies studying the action of PI 3-kinase inhibitors in a clinical setting. n We have provided lipid analysis for a number of external academic groups throughout 2018.

Publications www.babraham.ac.uk/science-services/biological-chemistry n Stark, A. K. et al. (2018) PI3Kδ hyper-activation promotes development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner. Nat. Commun. 9: 1-16 n Riento, K. et al. (2018) Flotillin proteins recruit sphingosine to membranes and maintain cellular sphingosine-1-phosphate levels. PLoS One 13: 1-18 n Chow, W. Y. et al. (2018) Essential but sparse collagen hydroxylysyl post-translational modifications detected by DNP NMR. Chem. Commun. 54: 12570-12573

45 Facilities

Tim Pearce Facility head Biological Support Unit

Facility members: 2 Deputy Facility Selected Impact Activities heads n A team of animal technicians and 7 Managers managers attended a LifeLab event held in Peterborough in 7 Supervisors September to promote careers in 3 Deputy Supervisors animal technology and engage 30 Experienced Animal members of the public in line with Technicians our commitment to the Concordat 4 Trainee/Apprentice on Openness on Animals in Animal Technicians Research. 3 Support Service n Facility tours and visits: All Technicians campus staff are invited to take 1 Administrator a virtual tour of the Institute’s animal facility to find out how our facility operates and how The use of animals in research continues researchers. We have a commitment to animals are used in our research. to be key in helping to understand uphold the highest standards of animal The facility ran three tours for biology and disease. The Biological welfare in all aspects of our work. campus staff in 2018. In addition, Support Unit provides state-of-the-art n Our Central Services unit utilises we continued to host visits from housing and care for pathogen-free robotic cage-washing technology and industry representatives, with rodents used in both academic and automated sterilisation processes to respect to animal facility design private company research programmes. provide equipment and consumables to for new builds and refurbishments, Our team of professionally qualified the bio-science units. and to share technical expertise animal technicians provide expert and guidance for their projects. technical support to researchers by Progress in 2018 undertaking regulated procedures, n In 2018, the BSU successfully formed n The 2018 KEC prize (and also the maintaining the animal health barrier a collaboration and partnership with Datesand group’s Janet Wood and undertaking animal husbandry. Agenda Resource Management creating Innovation Award) was awarded to a BSU Manager recognising their an ongoing recruitment initiative for Capabilities invention and development of apprentice animal technicians, with the n The BSU is made up of four bio-science an innovative animal enrichment BSU fast becoming acknowledged as units, each performing a unique role device. in the provision of flexible services to a centre-of-excellence for foundation meet the dynamic requirements of training and beyond. biological research. Our team of highly n The BSU continues to provide rentable trained animal technicians and service space and technical support to technicians perform daily animal commercial companies, with a 12% husbandry duties and provide essential increase in income from this venture services to the facility. compared to 2017. Throughout the year n Our animal technicians hold Home the facility has continued to draw a high Office Personal Licences enabling level of interest from new companies us to provide technical support for wanting to utilise the facility’s services.

www.babraham.ac.uk/science-services/biological-support-unit

46 Flow Cytometry

Flow cytometry is a powerful Capabilities Progress in 2018 technology allowing cells to be n State-of-the-art analysers: BD In 2018, the facility expanded with the identified, counted, analysed and LSRFortessa and Propel Lab YETI acquision of a new, high-end BD AriaFusion sorted on the basis of specific analysers allowing up to 30 parameters sorter to strengthen our cell sorting to be simultaneously analysed. capabilities. The facility also welcomed Rachael Walker physical or chemical features, two new members of staff to support the Facility head including using fluorescently labelled n Image cytometry: The facility houses sorting service. antibodies. The Flow Cytometry a Merck Millipore Imagestream MkII facility provides a world-class service allowing quantitative flow cytometry Facility members to enable the research goals of the data to be produced with images of Selected Impact Activities Flow cytometry specialist: Institute. We help scientists from both each cell. n Attila Bebes the Institute and external companies Flow cytometry training: n Cell sorting service: The facility provides Throughout 2018, the facility’s Rebecca Roberts with experimental design, training, an expert cell sorting service for Institute modular courses continued to troubleshooting and data analysis. Flow cytometry and external users. sell out with 240 delegates being technician: trained in 2018 (178 unique Isobel Darlington Aleksandra Lazowska delegates). Flow cytometry assistant: n The facility delivered bespoke flow Arthur Davis cytometry training at the Pirbright Institute.

Publications www.babraham.ac.uk/science-services/flow-cytometry @babraham_flow

47 Facilities

Our future services for this Gene Targeting & Genome Editing facility are currently under review. Introducing targeted changes to the mouse genome enables researchers to alter individual genes to study their functions. The Gene Targeting service is trialling new gene editing opportunities and technologies to produce genetically altered mouse models. Once up and running, the service will be able to aid in the design, generation, screening and evaluation of genetic modifications.

www.babraham.ac.uk/science-services/gene-targeting

48 Imaging

Simon Walker Facility head

Facility members Deputy manager: Hanneke Okkenhaug SEM specialist: Chieko Itakura (Started in 2018)

The Imaging facility provides a number n Image acquisition and data analysis for of services to support the Institute’s cell-based assays. Selected Impact Activities research. These include access to n Simon Walker presented the Progress in 2018 advanced light microscopy technologies, facility’s work at the first meeting 2018 has been a landmark year in the of BBSRC-funded imaging facilities. an electron microscopy service, and development of the Institute’s Imaging an advanced image analysis service. facility as we can now offer an electron n Hanneke Okkenhaug hosted two Training is a key part of our remit, microscopy capability to complement our GCSE school students on a week’s with users encouraged to learn how to advanced light microscopy resources. The work experience. operate our equipment independently, primary driver for this acquisition is a need n Simon Walker attended a LabLife with expert advice on hand to help when to study cellular ultrastructure at high event in Peterborough, sharing required. resolution in 3D, providing contextual STEM-related books that challenge information on organelle formation. Capabilities gender stereotypes with children. Installation of the microscope was n Scanning Electron Microscope with completed in April and a new member of Focussed Ion Beam (FIB SEM). staff joined our team in October to run n N-SIM/N-STORM super resolution the instrument and provide expertise on imaging system. sample preparation. We anticipate this resource to be fully operational in n Multi-photon microscope for intravital early 2019. imaging.

Publications www.babraham.ac.uk/science-services/imaging n Chrysanthou, S. et al. (2018). A critical role of TET1/2 proteins in cell-cycle progression of trophoblast stem cells. Stem Cell Rep. 10(4): 1355-1368 n White, M. A. et al. (2018). TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD. Nat. Neurosci. 21(4): 552-563 n Dautova, Y. et al. (2018). Calcium phosphate particles stimulate interleukin-1beta release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release. J Mol Cell Cardiol. 115: 82-93

49 Facilities

Lipidomics

Lipidomics identifies and quantifies the large array of lipid (fat) molecules found in cells, tissues and biological fluids. The lipidome is separated chromatographically and Michael Wakelam comprehensively characterised by Facility head high-accuracy and high-resolution tandem mass spectrometry. 15 novel Facility members chromatography methods have been Facility manager: developed, tested and validated to Andrea Lopez-Clavijo identify and semi-quantify a range of Postdoctoral research neutral, phospho-and sphingolipids with scientist: the aim of providing a detailed view of Aveline Neo the lipids in a biological system. This Research assistant: knowledge is then used to contribute Gregory West to an understanding of cell structures, signalling and regulation in a systems- wide investigation of metabolic changes in health and disease. Capabilities Progress in 2018 n The facility uses liquid chromatography We have successfully re-established Selected Impact Activities hyphenated to high resolution/high the Lipidomics facility adopting normal n Active collaborations with research mass accuracy mass spectrometry phase chromatography methods for the groups from the University for untargeted lipidomics (Orbitrap separation of phospholipids. The methods of Cambridge, King’s College technology). have been validated using targeted and London, Birmingham University, n Targeted lipidomics is performed untargeted approaches. In addition, Newfoundland University (Canada) by liquid chromatography or gas reverse phase chromatography has been and the University of Oxford. chromatography hyphenated to triple implemented for the separation of neutral n A service agreement for the quadrupole mass spectrometers. lipids. analysis of commercial samples by n Shotgun throughput analyses couples We are developing ion mobility methods the facility has been initiated with an Advion NanoMate to a high for the separation of ceramides with a company based in Oxford. resolution /high mass accuracy mass similar elemental composition coupled n Facility members promoted the spectrometer. with chromatography and targeted science carried out in the facility approaches. Methods to measure free fatty n Semi-quantitation of the lipids levels at the British Mass Spectrometry acids, cholesterol and cholesterol ester when compared to control samples Society annual conference. are also under development, to offer a prior to normalisation of the data to the comprehensive range of lipids to cover the weight/DNA/protein content. lipidome. Finally, we have introduced a fast n Hydrophobic extraction of the lipids and reliable cold homogenisation of soft present in cell lines and tissues from and bland tissue, including worms mouse, worm and human samples. (C. elegans) using a Precellys homogenizer.

Publications www.babraham.ac.uk/science-services/lipidomics n Sadej, R. et al. (2018). CD151 regulates expression of FGFR2 in breast cancer cells via PKC-dependent pathways. J Cell Sci. 131(21) n Burla, B. et al. (2018) MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines. J Lipid Res. 59(10): 2001-2017 n Nguyen, A. et al. (2018) Host lipidome analysis during rhinovirus replication in HBECs identifies potential therapeutic targets. J Lipid Res. 59(9): 1671-1684

50 Mass Spectrometry

Mass spectrometry is unrivalled in located in the Biochemistry n Quantitation of DNA modifications, its potential to identify, characterise department at the University of particularly cytosine modifications 5mC, and quantify almost any biological Cambridge. 5hmC, 5fC and 5caC. molecule, at very high sensitivity and in highly complex samples. The Mass n Full range of high-sensitivity mass n Development of novel mass David Oxley Spectrometry facility uses the latest spectrometric protein analyses spectrometric analytical methods. Facility head including: approaches and develops novel methods Progress in 2018 to analyse biological molecules, Facility members • quantitative proteome analysis During 2018 we worked with many of particularly proteins and nucleic acids, (label-free, SILAC, isobaric tagging); the Institute’s research groups on a range Senior research assistant: working with colleagues from across the of projects and also continued method Judith Webster institute and campus companies. • identification/quantitation of development. For example, in collaboration proteins in purified complexes; Postdoctoral researcher: Capabilities with ’s group, we extended our Katarzyna Wojdyla n Three high-resolution tandem mass • identification, localisation and methodology for the analysis of cytosine modifications in DNA to measure uracil, a spectrometers (Q Exactive Plus, Q quantitation of post-translational very rare base in genomic DNA, which may Exactive and Orbitrap Velos Pro) situated modifications; play a role in DNA demethylation. Working within the facility, and the facility has • detailed structural characterisation with the Kelsey lab, we used our low-level shared access to a state-of-the-art of individual proteins; TMT method for quantitative proteome Orbitrap Fusion Lumos instrument analysis of small numbers of mouse • targeted protein quantitation. oocytes. We also initiated a new project using a cell-surface labelling technique to investigate the cell-surface proteomes of naive vs primed human pluripotent stem cells (with Peter Rugg-Gunn).

Selected Impact Activities n Participated in the Institute’s ‘Race Against the Ageing Clock’ exhibit at the 2018 Royal Society Summer Science Exhibition in London (July). n Showcased the facility at the Babraham Research Campus science morning. n Commercial work for several Babraham Research Campus companies.

Publications www.babraham.ac.uk/science-services/mass-spectrometry n Tsolakos, N. et al. (2018) Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors. Proc Natl Acad Sci USA. 115(48):12176-12181 n Olova, N. et al. (2018) Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data. Genome Biol. 15;19(1):33 n Smyrnias, I. et al. (2018) Contractile responses to endothelin-1 are regulated by PKC phosphorylation of cardiac myosin binding protein-C in rat ventricular myocytes. J Mol Cell Cardiol. 117:1-18 51 Facilities

Next Generation Sequencing

SmartSeq v2 protocol and NEB Next Ultra II RNA-seq library preparation.

n Quality control services to ensure optimal yield and sequence quality. Kristina Tabbada Facility head n Coming in 2019 – automated single cell library preparation using the 10X Facility members Genomics Chromium Controller. Research assistant: Progress in 2018 Nicole Forrester The Next Generation Sequencing facility has continued to expand its range of services. In 2018, the facility began to offer automated RNA-seq library preparation services to make this powerful investigative tool available to a wider range of researchers. Library preparation using the Hamilton NGS Star liquid handling system with on-deck thermal cycling provides an integrated sample-to-sequence solution with enhanced reproducibility and throughput.

The 10X Genomics Chromium Controller single cell partitioning and barcoding system will allow researchers to study gene expression, copy number variation and chromatin accessibility as well as to profile the immune system repertoire at an unprecedented level of resolution.

Sequencing large amounts of DNA Capabilities from many samples – a process called n A range of sequencing instruments Selected Impact Activities high-throughput sequencing, has the (HiSeq 2500, NextSeq 500 and MiSeq) n Attended the Core Technologies potential to further our understanding enabling researchers to select the for Life Sciences (CTLS2018@VIB) of mechanisms for gene regulation. It sequencing depth and read length Conference, Ghent, Belgium, can also help to enhance our knowledge needed for their project. 1-4 July 2018. of DNA organisation and structure. The n Library preparation services using the Next Generation Sequencing (NGS) automated liquid handling technology facility provides researchers with access of the Hamilton NGS Star. Protocols to cutting-edge sequencing technology to advance their research. currently automated include the

Publications www.babraham.ac.uk/science-services/sequencing-facility n Cruz, C. et al. (2018) Tri-methylation of histone H3 lysine 4 facilitates gene expression in ageing cells. eLife 7: 34081 n Koohy, H. et al. (2018) Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells. Genome Biol. 19: 126 n Clark, S.C. et al. (2018) scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells Nat. Commun. 9: 781

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