Nature Protocols

Article	Citations
Tutorial: a guide to performing polygenic risk score analyses	893
The HDOCK server for integrated protein–protein docking	639
Using MetaboAnalyst 5.0 for LC–HRMS spectra processing, multi-omics integration and covariate adjustment of global metabolomics data	571
A scalable SCENIC workflow for single-cell gene regulatory network analysis	495
Reproducible molecular networking of untargeted mass spectrometry data using GNPS	338
Establishment of patient-derived cancer organoids for drug-screening applications	310
The trRosetta server for fast and accurate protein structure prediction	293
Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay	287
Tutorial: design and fabrication of nanoparticle-based lateral-flow immunoassays	238
CODEX multiplexed tissue imaging with DNA-conjugated antibodies	226
Design, fabrication and applications of tetrahedral DNA nanostructure-based multifunctional complexes in drug delivery and biomedical treatment	212
A practical guide to large-scale docking	209
Isolation and characterization of extracellular vesicle subpopulations from tissues	194
Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays	192
Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results	190
Efficient low-cost chromatin profiling with CUT&Tag	178
Tutorial: multivariate classification for vibrational spectroscopy in biological samples	177
Proximity labeling in mammalian cells with TurboID and split-TurboID	169
Tutorial: guidelines for the computational analysis of single-cell RNA sequencing data	167
Global analysis of repetitive DNA from unassembled sequence reads using RepeatExplorer2	145
Tutorial: guidelines for standardized performance tests for electrodes intended for neural interfaces and bioelectronics	142
MOF-derived nanoporous carbons with diverse tunable nanoarchitectures	131
Engineering SARS-CoV-2 using a reverse genetic system	131
Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography–mass spectrometry	129
Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development	125

Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles	120
Artificial intelligence–enabled virtual screening of ultra-large chemical libraries with deep docking	116
Tutorial: best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation	115
Optimization of metabolomic data processing using NOREVA	113
Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography	112
Metagenome analysis using the Kraken software suite	112
Standardized bacteriophage purification for personalized phage therapy	110
I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction	108
High-yield production of mono- or few-layer transition metal dichalcogenide nanosheets by an electrochemical lithium ion intercalation-based exfoliation method	104
Multiplexed single-cell proteomics using SCoPE2	104
Chromatin accessibility profiling by ATAC-seq	104
Generation of synthetic nanobodies against delicate proteins	103
Tutorial: guidelines for annotating single-cell transcriptomic maps using automated and manual methods	99
Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA	98
3D bioprinted silk fibroin hydrogels for tissue engineering	97
Intestinal organoid cocultures with microbes	97
Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids	96
Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery	94
High-throughput cultivation and identification of bacteria from the plant root microbiota	92
Precision genome editing using cytosine and adenine base editors in mammalian cells	92
Ligand binding free-energy calculations with funnel metadynamics	91
Jointly defining cell types from multiple single-cell datasets using LIGER	91
Algorithmic assessment of cellular senescence in experimental and clinical specimens	90
An in vitro batch fermentation protocol for studying the contribution of food to gut microbiota composition and functionality	89
Chemometric analysis in Raman spectroscopy from experimental design to machine learning–based modeling	89
Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing	88
A standardized social preference protocol for measuring social deficits in mouse models of autism	86
RNA-extraction-free nano-amplified colorimetric test for point-of-care clinical diagnosis of COVID-19	85
High-yield, green and scalable methods for producing MOF-303 for water harvesting from desert air	84
Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta	84
Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing	83
Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases	81
Measuring DNA modifications with the comet assay: a compendium of protocols	80
Engineered human blood–brain barrier microfluidic model for vascular permeability analyses	80
Microfluidic integration of regeneratable electrochemical affinity-based biosensors for continual monitoring of organ-on-a-chip devices	79
Analyzing high-dimensional cytometry data using FlowSOM	78
Cellular thermal shift assay for the identification of drug–target interactions in the Plasmodium falciparum proteome	75
Analysis of task-based functional MRI data preprocessed with fMRIPrep	75
Generation of insulin-producing pancreatic β cells from multiple human stem cell lines	73
A dynamic DNA tetrahedron framework for active targeting	73
Combined proximity labeling and affinity purification−mass spectrometry workflow for mapping and visualizing protein interaction networks	72
Establishment of human fetal hepatocyte organoids and CRISPR–Cas9-based gene knockin and knockout in organoid cultures from human liver	71
Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap	70
Rolling circle amplification (RCA)-based DNA hydrogel	70
A metabolic modeling platform for the computation of microbial ecosystems in time and space (COMETS)	69
Identifying unknown metabolites using NMR-based metabolic profiling techniques	69
3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert	67
Engineering brain assembloids to interrogate human neural circuits	64
Measuring the elastic modulus of soft culture surfaces and three-dimensional hydrogels using atomic force microscopy	64
Generic acquisition protocol for quantitative MRI of the spinal cord	63

lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements	63
The normative modeling framework for computational psychiatry	63
Improved high-molecular-weight DNA extraction, nanopore sequencing and metagenomic assembly from the human gut microbiome	62
Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging	62
Fabrication, characterization and applications of graphene electronic tattoos	61
Microtiter plate assays to assess antibiofilm activity against bacteria	59
Simultaneous noninvasive recording of electrocardiogram and skin sympathetic nerve activity (neuECG)	58
A robust unsupervised machine-learning method to quantify the morphological heterogeneity of cells and nuclei	56
Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks	55
Tools for experimental and computational analyses of off-target editing by programmable nucleases	55
Genetic circuit design automation with Cello 2.0	55
Accurate determination of protein:ligand standard binding free energies from molecular dynamics simulations	55
Synthesis of siRNA nanoparticles to silence plaque-destabilizing gene in atherosclerotic lesional macrophages	54
NovoSpaRc: flexible spatial reconstruction of single-cell gene expression with optimal transport	54
Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction	54
Solid-state 31P NMR mapping of active centers and relevant spatial correlations in solid acid catalysts	54
A well plate–based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature	54
Detection of aberrant gene expression events in RNA sequencing data	54
Synthesis of N-heterocyclic carbene gold(I) complexes	53
Digital postprocessing and image segmentation for objective analysis of colorimetric reactions	52
Tutorial: practical considerations for tissue clearing and imaging	52
Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope	52
Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH	52
Whole-brain functional ultrasound imaging in awake head-fixed mice	51
CRISPR–Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes	51
Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells	50
Tutorial: avoiding and correcting sample-induced spherical aberration artifacts in 3D fluorescence microscopy	49
Tutorial: using nanoneedles for intracellular delivery	49
Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq	48
A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents	47
Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity	47
Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae	47
Raman spectroscopy for real-time and in situ monitoring of mechanochemical milling reactions	46
Analysis of clonogenic growth in vitro	46
Stem-cell-derived human microglia transplanted into mouse brain to study human disease	45
Inducible mouse models of colon cancer for the analysis of sporadic and inflammation-driven tumor progression and lymph node metastasis	45
A universal approach for the synthesis of mesoporous gold, palladium and platinum films for applications in electrocatalysis	45
Comprehensive structural glycomic characterization of the glycocalyxes of cells and tissues	44
Characterization of connective tissues using near-infrared spectroscopy and imaging	44
Culture and analysis of kidney tubuloids and perfused tubuloid cells-on-a-chip	43
Cargo loading within ferritin nanocages in preparation for tumor-targeted delivery	43
Evaluation of the effects of cold atmospheric plasma and plasma-treated liquids in cancer cell cultures	43
Nanocarriers based on bacterial membrane materials for cancer vaccine delivery	43
CRISPR off-target detection with DISCOVER-seq	42
Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release	42
Use of deep learning to develop continuous-risk models for adverse event prediction from electronic health records	41
A complete and flexible workflow for metaproteomics data analysis based on MetaProteomeAnalyzer and Prophane	41
Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions	41
Mapping mechanical properties of biological materials via an add-on Brillouin module to confocal microscopes	41
Tutorial: a computational framework for the design and optimization of peripheral neural interfaces	41
COVIDep: a web-based platform for real-time reporting of vaccine target recommendations for SARS-CoV-2	41
Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq	41
Stable isotope tracing to assess tumor metabolism in vivo	41
Phage-assisted continuous and non-continuous evolution	40
Spooling electrochemiluminescence spectroscopy: development, applications and beyond	40
Transplantation of intestinal organoids into a mouse model of colitis	40
In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels	40
Design, assembly, and characterization of membrane-spanning DNA nanopores	40
Statistical analysis in metabolic phenotyping	39
A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility	39
IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues	38
Organotypic culture assays for murine and human primary and metastatic-site tumors	38
Design and simulation of DNA, RNA and hybrid protein–nucleic acid nanostructures with oxView	37
Chronic, cortex-wide imaging of specific cell populations during behavior	36
Use of human peripheral blood mononuclear cells to define immunological properties of nucleic acid nanoparticles	36
A generalized workflow for conducting electric field–optimized, fMRI-guided, transcranial magnetic stimulation	36
Complex-centric proteome profiling by SEC-SWATH-MS for the parallel detection of hundreds of protein complexes	35
Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry	35
A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes	35
Designing and executing prime editing experiments in mammalian cells	35
High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity	35
Composable microfluidic spinning platforms for facile production of biomimetic perfusable hydrogel microtubes	34
Tutorial: assessing metagenomics software with the CAMI benchmarking toolkit	34
Protein higher-order-structure determination by fast photochemical oxidation of proteins and mass spectrometry analysis	34
An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity	33
Expression and characterization of SARS-CoV-2 spike proteins	33
High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery	33
Smart-RRBS for single-cell methylome and transcriptome analysis	33
Engineering DNA nanostructures for siRNA delivery in plants	33
Macrophage phagocytosis assay with reconstituted target particles	32

A comprehensive guide to studying inflammasome activation and cell death	32
The maternal serum metabolome by multisegment injection-capillary electrophoresis-mass spectrometry: a high-throughput platform and standardized data workflow for large-scale epidemiological studies	32
Label-free capacitive assaying of biomarkers for molecular diagnostics	32
Subzero non-frozen preservation of human livers in the supercooled state	31
Integrative structural modeling of macromolecular complexes using Assembline	31
Responsive and activable nanomedicines for remodeling the tumor microenvironment	31
Selected ion flow tube mass spectrometry for targeted analysis of volatile organic compounds in human breath	31
Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture	31
Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues	31
Implantation of Neuropixels probes for chronic recording of neuronal activity in freely behaving mice and rats	31
Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures	31
Monomicellar assembly to synthesize structured and functional mesoporous carbonaceous nanomaterials	31
Graphene- and metal-induced energy transfer for single-molecule imaging and live-cell nanoscopy with (sub)-nanometer axial resolution	31
Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells	30
Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells	30
All-optical interrogation of neural circuits in behaving mice	30
Tutorial: structural characterization of isolated metal atoms and subnanometric metal clusters in zeolites	30
Quantification of organelle contact sites by split-GFP-based contact site sensors (SPLICS) in living cells	30
Imaging and optogenetic modulation of vascular mural cells in the live brain	30
Single-crystal structure determination of nanosized metal–organic frameworks by three-dimensional electron diffraction	29
Simple, efficient and thorough shotgun proteomic analysis with PatternLab V	29
Optimized single-nucleus transcriptional profiling by combinatorial indexing	29
The use of sonicated lipid vesicles for mass spectrometry of membrane protein complexes	29
Preparation and quantitative analysis of multicenter luminescence materials for sensing function	29
Effective high-throughput isolation of fully human antibodies targeting infectious pathogens	29
Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq	29
Coculture of primary human colon monolayer with human gut bacteria	29
Continuous human iPSC-macrophage mass production by suspension culture in stirred tank bioreactors	29
Perfusion and endothelialization of engineered tissues with patterned vascular networks	29
Spatial visualization of comprehensive brain neurotransmitter systems and neuroactive substances by selective in situ chemical derivatization mass spectrometry imaging	28
Testing physiologically relevant conditions in minimal inhibitory concentration assays	28
Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells	28
Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq	28
Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy	28
Implementation of a 4Pi-SMS super-resolution microscope	28
Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues	28
Large-scale site-specific mapping of the O-GalNAc glycoproteome	28
Nuclei isolation of multiple brain cell types for omics interrogation	27
Ex situ determination of freely dissolved concentrations of hydrophobic organic chemicals in sediments and soils: basis for interpreting toxicity and assessing bioavailability, risks and remediation n	27
Regional and functional division of functional elements of solid-state nanochannels for enhanced sensitivity and specificity of biosensing in complex matrices	27
Revealing nascent RNA processing dynamics with nano-COP	27
CRISPRi-seq for genome-wide fitness quantification in bacteria	27
Tracing DNA paths and RNA profiles in cultured cells and tissues with ORCA	27
Identification, isolation and analysis of human gut-associated lymphoid tissues	26
Creating hierarchical pores in metal–organic frameworks via postsynthetic reactions	26
Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue	26
Bacterial mock communities as standards for reproducible cytometric microbiome analysis	26
Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells	26
An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli	26
A methodological checklist for fMRI drug cue reactivity studies: development and expert consensus	26
Hyperosmolar blood–brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance	26
Determination of G-protein–coupled receptor oligomerization by molecular brightness analyses in single cells	26
Directed differentiation of human induced pluripotent stem cells to hepatic stellate cells	25
Nanoscale imaging of clinical specimens using conventional and rapid-expansion pathology	25
The cell-line-derived subcutaneous tumor model in preclinical cancer research	25
Proteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualization, identification and quantification of N-myristoylation, N- and S-acylation, O-cholesterylation, S-	25
Synthesis of high-molar-activity [18F]6-fluoro-l-DOPA suitable for human use via Cu-mediated fluorination of a BPin precursor	25
iBEAT V2.0: a multisite-applicable, deep learning-based pipeline for infant cerebral cortical surface reconstruction	25
Genetically modified mouse models to help fight COVID-19	25
Recovering prokaryotic genomes from host-associated, short-read shotgun metagenomic sequencing data	25
Precise motor mapping with transcranial magnetic stimulation	25
Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research	25
Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid	25
Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning	24
Considerations for implementing electronic laboratory notebooks in an academic research environment	24
GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos	24
Generation of heart-forming organoids from human pluripotent stem cells	24
Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture	24
In vivo compression and imaging in mouse brain to measure the effects of solid stress	24
Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables	24
Preparation of robust fluorescent probes for tracking endogenous formaldehyde in living cells and mouse tissue slices	24
Geometric morphometrics of microscopic animals as exemplified by model nematodes	24
FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing	24
SPRITE: a genome-wide method for mapping higher-order 3D interactions in the nucleus using combinatorial split-and-pool barcoding	24
Rapid in situ identification of biological specimens via DNA amplicon sequencing using miniaturized laboratory equipment	24
Preparation of near-infrared AIEgen-active fluorescent probes for mapping amyloid-β plaques in brain tissues and living mice	24
Probing low-copy-number proteins in single living cells using single-cell plasmonic immunosandwich assays	23
Transcription factor chromatin profiling genome-wide using uliCUT&RUN in single cells and individual blastocysts	23
Scalable characterization of the PAM requirements of CRISPR–Cas enzymes using HT-PAMDA	23
Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain	23
A simple sperm-sexing method that activates TLR7/8 on X sperm for the efficient production of sexed mouse or cattle embryos	23
High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening	23
Simultaneous quantification of protein–DNA interactions and transcriptomes in single cells with scDam&T-seq	23
Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping	22
A graphical user interface to design high-throughput optogenetic experiments with the optoPlate-96	22
Raman spectral cytopathology for cancer diagnostic applications	22
Isotropic imaging across spatial scales with axially swept light-sheet microscopy	22
Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2)	22
Synthesis and characterization of chemically fueled supramolecular materials driven by carbodiimide-based fuels	22
Decoding personal biotic and abiotic airborne exposome	22
Utilizing Skyline to analyze lipidomics data containing liquid chromatography, ion mobility spectrometry and mass spectrometry dimensions	22
Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria	22
Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases	22
Assessing motor-related phenotypes of Caenorhabditis elegans with the wide field-of-view nematode tracking platform	22
Comprehensive isolation of extracellular vesicles and nanoparticles	22