Protocols

  • Protocol |

    This protocol describes how to create functional chromosome fusions in yeast through sequential rounds of CRISPR–Cas9-guided homologous recombination. Each round of pairwise chromosome end-to-end fusion deletes two telomeres and one centromere.

    • Yangyang Shao
    • , Ning Lu
    • , Xiaoli Xue
    •  & Zhongjun Qin
  • Protocol |

    A protocol for the design, construction, and operation of an intelligent image-activated cell sorting (iIACS) machine that performs real-time image-based sorting of single cells from heterogeneous populations with high throughput and intelligence.

    • Akihiro Isozaki
    • , Hideharu Mikami
    • , Kotaro Hiramatsu
    • , Shinya Sakuma
    • , Yusuke Kasai
    • , Takanori Iino
    • , Takashi Yamano
    • , Atsushi Yasumoto
    • , Yusuke Oguchi
    • , Nobutake Suzuki
    • , Yoshitaka Shirasaki
    • , Taichiro Endo
    • , Takuro Ito
    • , Kei Hiraki
    • , Makoto Yamada
    • , Satoshi Matsusaka
    • , Takeshi Hayakawa
    • , Hideya Fukuzawa
    • , Yutaka Yatomi
    • , Fumihito Arai
    • , Dino Di Carlo
    • , Atsuhiro Nakagawa
    • , Yu Hoshino
    • , Yoichiroh Hosokawa
    • , Sotaro Uemura
    • , Takeaki Sugimura
    • , Yasuyuki Ozeki
    • , Nao Nitta
    •  & Keisuke Goda
  • Protocol |

    This protocol details labeling of bacterial outer-membrane proteins with spin labels to study conformational changes and their interaction with ligands and substrates in situ, using pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy.

    • Benesh Joseph
    • , Eva A. Jaumann
    • , Arthur Sikora
    • , Katja Barth
    • , Thomas F. Prisner
    •  & David S. Cafiso
  • Protocol |

    Cross-linking with mass spectrometry (XL-MS) can reveal the topology of protein complexes. This protocol describes how to synthesize a cleavable cross-linker and use it to map protein structures and interactions within intact cells and animal tissues.

    • Juan D. Chavez
    • , Jared P. Mohr
    • , Martin Mathay
    • , Xuefei Zhong
    • , Andrew Keller
    •  & James E. Bruce
  • Protocol |

    DNA origami can be used to create a wide diversity of 3D structures. This protocol describes a DNA origami silicification (DOS) approach for generating complex silica composite nanomaterials with tailored structural properties.

    • Xinxin Jing
    • , Fei Zhang
    • , Muchen Pan
    • , Xinpei Dai
    • , Jiang Li
    • , Lihua Wang
    • , Xiaoguo Liu
    • , Hao Yan
    •  & Chunhai Fan
  • Protocol |

    The transmembrane domains of many type I/II membrane proteins oligomerize in the lipid bilayer, mediating protein assembly and clustering that are critical to their function. The authors provide a general protocol for determining the structures of these domains in a near–lipid bilayer environment.

    • Qingshan Fu
    • , Alessandro Piai
    • , Wen Chen
    • , Ke Xia
    •  & James J. Chou
  • Protocol |

    A protocol for the assembly and use of the optoPlate-96, a platform for high-throughput three-color optogenetics experiments in microwell plates. With the provided design files, users can assemble the optoPlate-96 from 3D-printed and laser-cut components.

    • Lukasz J. Bugaj
    •  & Wendell A. Lim
  • Protocol |

    This protocol outlines the chemical synthesis of proteins via a recently discovered reaction called diselenide–selenoester ligation (DSL) in a rapid, additive-free manner. After ligation, products can be chemoselectively deselenized to produce native peptide and protein products.

    • Sameer S. Kulkarni
    • , Emma E. Watson
    • , Bhavesh Premdjee
    • , Kilian W. Conde-Frieboes
    •  & Richard J. Payne
  • Protocol |

    This protocol describes how to use an open-source toolbox, DeepLabCut, to train a deep neural network to precisely track user-defined features with limited training data. This allows noninvasive behavioral tracking of movement.

    • Tanmay Nath
    • , Alexander Mathis
    • , An Chi Chen
    • , Amir Patel
    • , Matthias Bethge
    •  & Mackenzie Weygandt Mathis
  • Protocol |

    Here, the authors describe the use of synthetic DNA spike-ins, called sequins, as internal quantitative and qualitative controls throughout the sequencing workflow. They detail their application in whole-genome and targeted human genome sequencing.

    • James Blackburn
    • , Ted Wong
    • , Bindu Swapna Madala
    • , Chris Barker
    • , Simon A. Hardwick
    • , Andre L. M. Reis
    • , Ira W. Deveson
    •  & Tim R. Mercer
  • Protocol |

    This protocol describes a solvent-assisted method to prepare supported lipid bilayers (SLBs) with minimal sample preparation. The approach is quick, easy to implement, and compatible with a wide range of lipid compositions and material supports.

    • Abdul Rahim Ferhan
    • , Bo Kyeong Yoon
    • , Soohyun Park
    • , Tun Naw Sut
    • , Hokyun Chin
    • , Jae Hyeon Park
    • , Joshua A. Jackman
    •  & Nam-Joon Cho
  • Protocol |

    This protocol describes the steps in GRID-seq, a procedure used to capture RNA in the proximity of chromatin in situ, thus enabling systematic detection of RNA–chromatin interactions in mammalian genomes.

    • Bing Zhou
    • , Xiao Li
    • , Daji Luo
    • , Do-Hwan Lim
    • , Yu Zhou
    •  & Xiang-Dong Fu
  • Protocol |

    This protocol describes how to fabricate and use a wireless nanopore electrode (WNE). The WNE has metal at the tip of a nanopipette, which acts as an electroactive sensing interface, providing electrochemical measurements of single entities.

    • Rui Gao
    • , Yao Lin
    • , Yi-Lun Ying
    • , Yong-Xu Hu
    • , Su-Wen Xu
    • , Lin-Qi Ruan
    • , Ru-Jia Yu
    • , Yuan-Jie Li
    • , Hao-Wen Li
    • , Ling-Fei Cui
    •  & Yi-Tao Long
  • Protocol |

    This protocol describes how to derive and maintain mouse haploid embryonic stem cells (hESCs) from female gametes. Additional procedures that can be carried out with cell lines obtained from the mouse cell resource Haplobank are also described.

    • Ulrich Elling
    • , Michael Woods
    • , Josep V. Forment
    • , Beiyuan Fu
    • , Fengtang Yang
    • , Bee Ling Ng
    • , Jose R. Vicente
    • , David J. Adams
    • , Brendan Doe
    • , Stephen P. Jackson
    • , Josef M. Penninger
    •  & Gabriel Balmus
  • Protocol |

    This protocol describes the design, application and computational analysis of high-dimensional fluorescent antibody panels for flow cytometry. Up to 28 colors can be characterized to study complex cellular populations such as the immune system.

    • Jolanda Brummelman
    • , Claudia Haftmann
    • , Nicolás Gonzalo Núñez
    • , Giorgia Alvisi
    • , Emilia M. C. Mazza
    • , Burkhard Becher
    •  & Enrico Lugli
  • Protocol |

    Bioengineered biliary tissue suitable for biliary reconstruction is obtained from cholangiocyte organoids derived from human primary extra- or intrahepatic duct cholangiocytes.

    • Olivia C. Tysoe
    • , Alexander W. Justin
    • , Teresa Brevini
    • , Si Emma Chen
    • , Krishnaa T. Mahbubani
    • , Anna K. Frank
    • , Hajer Zedira
    • , Espen Melum
    • , Kourosh Saeb-Parsy
    • , Athina E. Markaki
    • , Ludovic Vallier
    •  & Fotios Sampaziotis
  • Protocol |

    Pei et al. describe a genetic barcoding system termed Polylox. Induced by transient activity of Cre recombinase in specific mouse tissues, random DNA recombination in the Polylox substrate produces unique barcodes that can be used for fate mapping.

    • Weike Pei
    • , Xi Wang
    • , Jens Rössler
    • , Thorsten B. Feyerabend
    • , Thomas Höfer
    •  & Hans-Reimer Rodewald
  • Protocol |

    The percentage of cancer neoantigens that are spontaneously recognized by T cells is generally very low. This protocol describes how CD8+ T cells from healthy donors can be used for enhanced targeting of these neoantigens.

    • Muhammad Ali
    • , Zsofia Foldvari
    • , Eirini Giannakopoulou
    • , Maxi-Lu Böschen
    • , Erlend Strønen
    • , Weiwen Yang
    • , Mireille Toebes
    • , Benjamin Schubert
    • , Oliver Kohlbacher
    • , Ton N. Schumacher
    •  & Johanna Olweus
  • Protocol |

    This protocol describes the design and fabrication of nanoscale structures for studying intracellular responses to membrane curvature, which are visualized by fluorescence imaging; FIB–SEM is used to characterize the nanostructure–cell interface.

    • Xiao Li
    • , Laura Matino
    • , Wei Zhang
    • , Lasse Klausen
    • , Allister F. McGuire
    • , Claudia Lubrano
    • , Wenting Zhao
    • , Francesca Santoro
    •  & Bianxiao Cui
  • Protocol |

    The design of proteins that can be controlled by rapamycin or by light is discussed, as well as how to identify sites for insertion of engineered regulatory domains and test the analogs biochemically and in living cells.

    • Onur Dagliyan
    • , Nikolay V. Dokholyan
    •  & Klaus M. Hahn
  • Protocol |

    This protocol for clearing and high-resolution 3D imaging of entire organoids expressing fluorescence reporters or following immunolabeling enables confocal, super-resolution confocal, multiphoton and light-sheet microscopy to be performed.

    • Johanna F. Dekkers
    • , Maria Alieva
    • , Lianne M. Wellens
    • , Hendrikus C. R. Ariese
    • , Paul R. Jamieson
    • , Annelotte M. Vonk
    • , Gimano D. Amatngalim
    • , Huili Hu
    • , Koen C. Oost
    • , Hugo J. G. Snippert
    • , Jeffrey M. Beekman
    • , Ellen J. Wehrens
    • , Jane E. Visvader
    • , Hans Clevers
    •  & Anne C. Rios
  • Protocol |

    This protocol is used to detect RNA decay intermediates in human cultured cells. Reporter mRNAs can be detected and quantified with molecular biology (northern blot, qPCR) and single-molecule fluorescence microscopy (smFISH, live imaging) approaches.

    • Franka Voigt
    • , Jennifer V. Gerbracht
    • , Volker Boehm
    • , Ivana Horvathova
    • , Jan Eglinger
    • , Jeffrey A. Chao
    •  & Niels H. Gehring
  • Protocol |

    This protocol describes the design, in vitro characterization, and imaging applications of iGluSnFR-based genetically encoded glutamate indicators (GEGIs) in tissue culture of rat hippocampus.

    • Céline D. Dürst
    • , J. Simon Wiegert
    • , Nordine Helassa
    • , Silke Kerruth
    • , Catherine Coates
    • , Christian Schulze
    • , Michael A. Geeves
    • , Katalin Török
    •  & Thomas G. Oertner
  • Protocol |

    18F labeling of non-activated arenes (e.g., 3-[18F]fluoro-5-[(pyridin-3-yl)ethynyl] benzonitrile ([18F]FPEB)) is an unmet need for PET imaging. This protocol uses a spirocyclic iodonium ylide method for one-step, regioselective radiofluorination.

    • Steven H. Liang
    • , Lu Wang
    • , Nickeisha A. Stephenson
    • , Benjamin H. Rotstein
    •  & Neil Vasdev
  • Protocol |

    This protocol describes how to fabricate and apply silicon-based structures for optically controlled neuromodulation. The structures can be used for nongenetic neuronal excitation in cultured neurons, brain slices, and in vivo applications.

    • Yuanwen Jiang
    • , Ramya Parameswaran
    • , Xiaojian Li
    • , João L. Carvalho-de-Souza
    • , Xiang Gao
    • , Lingyuan Meng
    • , Francisco Bezanilla
    • , Gordon M. G. Shepherd
    •  & Bozhi Tian
  • Protocol |

    This protocol provides instructions for using sigQC to obtain a set of quality control metrics to aid in the evaluation of gene signatures across datasets.

    • Andrew Dhawan
    • , Alessandro Barberis
    • , Wei-Chen Cheng
    • , Enric Domingo
    • , Catharine West
    • , Tim Maughan
    • , Jacob G. Scott
    • , Adrian L. Harris
    •  & Francesca M. Buffa
  • Protocol |

    A new protocol for high-speed whole-brain fluorescence imaging using block-face serial microscopy tomography (FAST). The protocol contains procedures for hardware assembly, sample preparation, imaging and data processing.

    • Kaoru Seiriki
    • , Atsushi Kasai
    • , Takanobu Nakazawa
    • , Misaki Niu
    • , Yuichiro Naka
    • , Masato Tanuma
    • , Hisato Igarashi
    • , Kosei Yamaura
    • , Atsuko Hayata-Takano
    • , Yukio Ago
    •  & Hitoshi Hashimoto
  • Protocol |

    Mouse pluripotent stem cells are grown in vitro into embryoid bodies before transplantation into mice, where they further differentiate into an integumentary organ system with appendages, including hair follicles and sebaceous glands.

    • Koh-ei Toyoshima
    • , Miho Ogawa
    •  & Takashi Tsuji
  • Protocol |

    The aim of this spectral standardization model is to expedite multicenter studies with large numbers of samples. The protocol covers sample preparation, acquisition of FTIR spectra, data preprocessing and model standardization.

    • Camilo L. M. Morais
    • , Maria Paraskevaidi
    • , Li Cui
    • , Nigel J. Fullwood
    • , Martin Isabelle
    • , Kássio M. G. Lima
    • , Pierre L. Martin-Hirsch
    • , Hari Sreedhar
    • , Júlio Trevisan
    • , Michael J. Walsh
    • , Dayi Zhang
    • , Yong-Guan Zhu
    •  & Francis L. Martin
  • Protocol |

    This protocol describes how to perform nanoscale chemical imaging using tip-enhanced Raman spectroscopy (TERS). The procedure details the preparation of plasmonically active TERS probes, alignment of a TERS system, and various example procedures.

    • Naresh Kumar
    • , Bert M. Weckhuysen
    • , Andrew J. Wain
    •  & Andrew J. Pollard
  • Protocol |

    This protocol describes the fabrication and practical applications of molybdenum disulfide (MoS2) nanopores. The procedure contains different methods for the transfer of monolayer MoS2, nanopore creation, and data acquisition and analysis.

    • Michael Graf
    • , Martina Lihter
    • , Mukeshchand Thakur
    • , Vasileia Georgiou
    • , Juraj Topolancik
    • , B. Robert Ilic
    • , Ke Liu
    • , Jiandong Feng
    • , Yann Astier
    •  & Aleksandra Radenovic