Table of contents

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The past, present and future of microbiome analyses pp2049 - 2053

Richard Allen White III, Stephen J Callister, Ronald J Moore, Erin S Baker & Janet K Jansson


This Perspective from Janet Jansson and colleagues reviews the development of microbiome analysis technologies over the last decade, and comments on the future potential of this fast-moving field.



Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION pp2054 - 2065

Tanmay A M Bharat & Sjors H W Scheres


Electron cryo-tomography produces 3D pictures of unique objects such as viruses, organelles or cells. This is a protocol for macromolecular structure determination from tomographic data using subtomogram averaging in the RELION software.

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy pp2066 - 2080

Richard N Day, Wen Tao & Kenneth W Dunn


This protocol describes a simple pipeline for imaging FRET biosensor probes with two-photon laser scanning microscopy (TPLSM). Validated FRET standards are described that can be used with any TPLSM setup to ensure sensitive detection of FRET ratios.

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq pp2081 - 2103

Iain C Macaulay, Mabel J Teng, Wilfried Haerty, Parveen Kumar, Chris P Ponting & Thierry Voet


G&T-seq enables sequencing of DNA and mRNA from the same single cell, allowing the effects of genomic variation on transcription to be studied. It is compatible with any WGA method and so can be tailored to specific applications.

Mapping 3D genome architecture through in situ DNase Hi-C pp2104 - 2121

Vijay Ramani, Darren A Cusanovich, Ronald J Hause, Wenxiu Ma, Ruolan Qiu, Xinxian Deng, C Anthony Blau, Christine M Disteche, William S Noble, Jay Shendure & Zhijun Duan


Ramani et al. describe a protocol for in situ DNase Hi-C as an alternative to traditional Hi-C methods that use restriction enzymes. The use of DNase I for chromatin digestion circumvents the resolution limit imposed when relying on genomic restriction sites.

Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments pp2122 - 2153

Cy M Jeffries, Melissa A Graewert, Clément E Blanchet, David B Langley, Andrew E Whitten & Dmitri I Svergun


Small-angle X-ray and neutron scattering are used to extract structural parameters and derive structural models of macromolecules in solution. The preparation of pure, monodisperse samples and exactly matching solvent blanks is crucial to the experiments' success.

Modeling and correction of structural variations in patient-derived iPSCs using CRISPR/Cas9 pp2154 - 2169

Chul-Yong Park, Jin Jea Sung, Sang-Hwi Choi, Dongjin R Lee, In-Hyun Park & Dong-Wook Kim


This protocol extends the use of genome editing technology to the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions. The authors use the CRISPR/Cas system to edit human induced pluripotent stem cells.

Remodeling somatic nuclei via exogenous expression of protamine 1 to create spermatid-like structures for somatic nuclear transfer pp2170 - 2188

Marta Czernik, Domenico Iuso, Paola Toschi, Saadi Khochbin & Pasqualino Loi


This protocol describes how to remodel sheep and mouse fibroblast nuclei into spermatid-like structures by exogenous expression of the protamine 1 gene. Blastocyst formation following somatic cell nuclear transfer in sheep oocytes is also described.

Serial sequencing of isolength RAD tags for cost-efficient genome-wide profiling of genetic and epigenetic variations pp2189 - 2200

Shi Wang, Pingping Liu, Jia Lv, Yangping Li, Taoran Cheng, Lingling Zhang, Yu Xia, Hongzhen Sun, Xiaoli Hu & Zhenmin Bao


This protocol uses restriction enzymes to produce isolength tags that are concatenated and sequenced to allow genome-wide genotyping and quantification of DNA methylation levels.

Fabrication and utility of a transparent graphene neural electrode array for electrophysiology, in vivo imaging, and optogenetics pp2201 - 2222

Dong-Wook Park, Sarah K Brodnick, Jared P Ness, Farid Atry, Lisa Krugner-Higby, Amelia Sandberg, Solomon Mikael, Thomas J Richner, Joseph Novello, Hyungsoo Kim, Dong-Hyun Baek, Jihye Bong, Seth T Frye, Sanitta Thongpang, Kyle I Swanson, Wendell Lake, Ramin Pashaie, Justin C Williams & Zhenqiang Ma


The protocol describes how to make transparent graphene neural electrodes for implantation onto the surface of the cerebral cortex in rodents and subsequent neural analysis by fluorescence microscopy, electrophysiology, optical coherence tomography, and optogenetics.

Self-organization of human embryonic stem cells on micropatterns pp2223 - 2232

Alessia Deglincerti, Fred Etoc, M Cecilia Guerra, Iain Martyn, Jakob Metzger, Albert Ruzo, Mijo Simunovic, Anna Yoney, Ali H Brivanlou, Eric Siggia & Aryeh Warmflash


This protocol describes how to differentiate and image human embryonic stem cells on micropatterned colonies to create radially organized domains of the germ layers mimicking embryonic gastrulation in vitro.

The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases pp2233 - 2242

Matthias Schmitz, Maria Cramm, Franc Llorens, Dominik Müller-Cramm, Steven Collins, Ryuichiro Atarashi, Katsuya Satoh, Christina D Orrù, Bradley R Groveman, Saima Zafar, Walter J Schulz-Schaeffer, Byron Caughey & Inga Zerr


This 96-well-plate ‘real-time quaking-induced conversion’ assay allows the detection of abnormal prion protein in human brain and CSF samples. It can be applied to study many protein misfolding diseases, as well as for drug screening and prion strain discrimination.

Assembly of multienzyme complexes on DNA nanostructures pp2243 - 2273

Jinglin Fu, Yuhe Renee Yang, Soma Dhakal, Zhao Zhao, Minghui Liu, Ting Zhang, Nils G Walter & Hao Yan


This protocol from Fu et al. describes the use of self-assembled DNA nanostructures as assembly scaffolds to spatially arrange multienzyme complexes and probe their interactions.

Identification and propagation of haploid human pluripotent stem cells pp2274 - 2286

Ido Sagi, Dieter Egli & Nissim Benvenisty


This protocol describes how to derive haploid human embryonic stem cells (ESCs) by parthenogenesis, and how to isolate and maintain them by cell sorting. Haploid human ESCs have a wide differentiation potential and are useful for genetic screening.

Preparation of plasmonic vesicles from amphiphilic gold nanocrystals grafted with polymer brushes pp2287 - 2299

Jibin Song, Peng Huang & Xiaoyuan Chen


This protocol describes the design and synthesis of amphiphilic gold nanocrystals coated with polymer brushes and their self-assembly into plasmonic vesicles.


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