Nature Protocols

Article	Citations
Tutorial: a guide to performing polygenic risk score analyses	919
Using MetaboAnalyst 5.0 for LC–HRMS spectra processing, multi-omics integration and covariate adjustment of global metabolomics data	610
A scalable SCENIC workflow for single-cell gene regulatory network analysis	519
Reproducible molecular networking of untargeted mass spectrometry data using GNPS	354
Establishment of patient-derived cancer organoids for drug-screening applications	315
The trRosetta server for fast and accurate protein structure prediction	308
Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay	295
Tutorial: design and fabrication of nanoparticle-based lateral-flow immunoassays	246
CODEX multiplexed tissue imaging with DNA-conjugated antibodies	239
A practical guide to large-scale docking	222
Design, fabrication and applications of tetrahedral DNA nanostructure-based multifunctional complexes in drug delivery and biomedical treatment	221
Isolation and characterization of extracellular vesicle subpopulations from tissues	204
Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results	198
Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays	197
Efficient low-cost chromatin profiling with CUT&Tag	186
Tutorial: multivariate classification for vibrational spectroscopy in biological samples	185
Proximity labeling in mammalian cells with TurboID and split-TurboID	180
Tutorial: guidelines for the computational analysis of single-cell RNA sequencing data	177
Global analysis of repetitive DNA from unassembled sequence reads using RepeatExplorer2	154
Tutorial: guidelines for standardized performance tests for electrodes intended for neural interfaces and bioelectronics	147
MOF-derived nanoporous carbons with diverse tunable nanoarchitectures	139
Metagenome analysis using the Kraken software suite	136
Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography–mass spectrometry	134
Engineering SARS-CoV-2 using a reverse genetic system	134
Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development	127

Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles	125
Artificial intelligence–enabled virtual screening of ultra-large chemical libraries with deep docking	123
Tutorial: best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation	122
I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction	118
Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography	116
Optimization of metabolomic data processing using NOREVA	114
Standardized bacteriophage purification for personalized phage therapy	112
Chromatin accessibility profiling by ATAC-seq	112
Multiplexed single-cell proteomics using SCoPE2	110
Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery	109
High-yield production of mono- or few-layer transition metal dichalcogenide nanosheets by an electrochemical lithium ion intercalation-based exfoliation method	107
Tutorial: guidelines for annotating single-cell transcriptomic maps using automated and manual methods	105
3D bioprinted silk fibroin hydrogels for tissue engineering	104
Intestinal organoid cocultures with microbes	101
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	101
An in vitro batch fermentation protocol for studying the contribution of food to gut microbiota composition and functionality	99
Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids	99
Chemometric analysis in Raman spectroscopy from experimental design to machine learning–based modeling	98
High-throughput cultivation and identification of bacteria from the plant root microbiota	96
Precision genome editing using cytosine and adenine base editors in mammalian cells	96
Algorithmic assessment of cellular senescence in experimental and clinical specimens	95
Ligand binding free-energy calculations with funnel metadynamics	93
Jointly defining cell types from multiple single-cell datasets using LIGER	92
A standardized social preference protocol for measuring social deficits in mouse models of autism	91
High-yield, green and scalable methods for producing MOF-303 for water harvesting from desert air	90
RNA-extraction-free nano-amplified colorimetric test for point-of-care clinical diagnosis of COVID-19	89
Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing	89
Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases	87
Measuring DNA modifications with the comet assay: a compendium of protocols	86
Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta	85
Engineered human blood–brain barrier microfluidic model for vascular permeability analyses	83
Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing	83
Analysis of task-based functional MRI data preprocessed with fMRIPrep	83
Microfluidic integration of regeneratable electrochemical affinity-based biosensors for continual monitoring of organ-on-a-chip devices	83
Analyzing high-dimensional cytometry data using FlowSOM	81
Rolling circle amplification (RCA)-based DNA hydrogel	77
Generation of insulin-producing pancreatic β cells from multiple human stem cell lines	77
A dynamic DNA tetrahedron framework for active targeting	75
Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap	74
Combined proximity labeling and affinity purification−mass spectrometry workflow for mapping and visualizing protein interaction networks	73
A metabolic modeling platform for the computation of microbial ecosystems in time and space (COMETS)	72
Establishment of human fetal hepatocyte organoids and CRISPR–Cas9-based gene knockin and knockout in organoid cultures from human liver	72
3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert	71
Identifying unknown metabolites using NMR-based metabolic profiling techniques	70
Generic acquisition protocol for quantitative MRI of the spinal cord	67
Measuring the elastic modulus of soft culture surfaces and three-dimensional hydrogels using atomic force microscopy	67
Engineering brain assembloids to interrogate human neural circuits	67
Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging	66
The normative modeling framework for computational psychiatry	66
lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements	65

Microtiter plate assays to assess antibiofilm activity against bacteria	65
Improved high-molecular-weight DNA extraction, nanopore sequencing and metagenomic assembly from the human gut microbiome	64
Fabrication, characterization and applications of graphene electronic tattoos	64
A robust unsupervised machine-learning method to quantify the morphological heterogeneity of cells and nuclei	58
NovoSpaRc: flexible spatial reconstruction of single-cell gene expression with optimal transport	58
Accurate determination of protein:ligand standard binding free energies from molecular dynamics simulations	57
Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks	56
Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction	56
Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH	56
Detection of aberrant gene expression events in RNA sequencing data	56
Tools for experimental and computational analyses of off-target editing by programmable nucleases	56
Genetic circuit design automation with Cello 2.0	56
Whole-brain functional ultrasound imaging in awake head-fixed mice	55
Digital postprocessing and image segmentation for objective analysis of colorimetric reactions	55
A well plate–based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature	55
Tutorial: practical considerations for tissue clearing and imaging	55
Synthesis of siRNA nanoparticles to silence plaque-destabilizing gene in atherosclerotic lesional macrophages	54
Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope	54
Solid-state 31P NMR mapping of active centers and relevant spatial correlations in solid acid catalysts	54
CRISPR–Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes	53
Synthesis of N-heterocyclic carbene gold(I) complexes	53
A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents	52
Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae	51
Tutorial: avoiding and correcting sample-induced spherical aberration artifacts in 3D fluorescence microscopy	51
Tutorial: using nanoneedles for intracellular delivery	50
Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells	50
Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity	49
Stem-cell-derived human microglia transplanted into mouse brain to study human disease	48
Analysis of clonogenic growth in vitro	48
Nanocarriers based on bacterial membrane materials for cancer vaccine delivery	47
Comprehensive structural glycomic characterization of the glycocalyxes of cells and tissues	47
Cargo loading within ferritin nanocages in preparation for tumor-targeted delivery	47
Raman spectroscopy for real-time and in situ monitoring of mechanochemical milling reactions	47
A universal approach for the synthesis of mesoporous gold, palladium and platinum films for applications in electrocatalysis	47
Inducible mouse models of colon cancer for the analysis of sporadic and inflammation-driven tumor progression and lymph node metastasis	45
Evaluation of the effects of cold atmospheric plasma and plasma-treated liquids in cancer cell cultures	45
Stable isotope tracing to assess tumor metabolism in vivo	44
A complete and flexible workflow for metaproteomics data analysis based on MetaProteomeAnalyzer and Prophane	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	44
Mapping mechanical properties of biological materials via an add-on Brillouin module to confocal microscopes	44
Transplantation of intestinal organoids into a mouse model of colitis	43
Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq	43
Tutorial: a computational framework for the design and optimization of peripheral neural interfaces	43
Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release	43
IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues	42
Use of deep learning to develop continuous-risk models for adverse event prediction from electronic health records	42
Statistical analysis in metabolic phenotyping	41
Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions	41
COVIDep: a web-based platform for real-time reporting of vaccine target recommendations for SARS-CoV-2	41
Design and simulation of DNA, RNA and hybrid protein–nucleic acid nanostructures with oxView	40
Design, assembly, and characterization of membrane-spanning DNA nanopores	40
Phage-assisted continuous and non-continuous evolution	40
Spooling electrochemiluminescence spectroscopy: development, applications and beyond	40
In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels	40
Designing and executing prime editing experiments in mammalian cells	39
A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility	39
Organotypic culture assays for murine and human primary and metastatic-site tumors	38
Chronic, cortex-wide imaging of specific cell populations during behavior	38
A generalized workflow for conducting electric field–optimized, fMRI-guided, transcranial magnetic stimulation	38
Use of human peripheral blood mononuclear cells to define immunological properties of nucleic acid nanoparticles	37
High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity	37
Complex-centric proteome profiling by SEC-SWATH-MS for the parallel detection of hundreds of protein complexes	37
Composable microfluidic spinning platforms for facile production of biomimetic perfusable hydrogel microtubes	37
Tutorial: assessing metagenomics software with the CAMI benchmarking toolkit	36
Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues	35
An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity	35
A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes	35
Spatially resolved analysis of FFPE tissue proteomes by quantitative mass spectrometry	35
Protein higher-order-structure determination by fast photochemical oxidation of proteins and mass spectrometry analysis	34
The maternal serum metabolome by multisegment injection-capillary electrophoresis-mass spectrometry: a high-throughput platform and standardized data workflow for large-scale epidemiological studies	34
Engineering DNA nanostructures for siRNA delivery in plants	34
All-optical interrogation of neural circuits in behaving mice	34
Expression and characterization of SARS-CoV-2 spike proteins	34
Quantification of organelle contact sites by split-GFP-based contact site sensors (SPLICS) in living cells	34
Macrophage phagocytosis assay with reconstituted target particles	34
A comprehensive guide to studying inflammasome activation and cell death	33
Monomicellar assembly to synthesize structured and functional mesoporous carbonaceous nanomaterials	33
Implantation of Neuropixels probes for chronic recording of neuronal activity in freely behaving mice and rats	33
Coculture of primary human colon monolayer with human gut bacteria	33

High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery	33
Optimized single-nucleus transcriptional profiling by combinatorial indexing	33
Smart-RRBS for single-cell methylome and transcriptome analysis	33
Responsive and activable nanomedicines for remodeling the tumor microenvironment	33
Label-free capacitive assaying of biomarkers for molecular diagnostics	32
Continuous human iPSC-macrophage mass production by suspension culture in stirred tank bioreactors	32
Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture	32
Creating hierarchical pores in metal–organic frameworks via postsynthetic reactions	32
Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures	32
Graphene- and metal-induced energy transfer for single-molecule imaging and live-cell nanoscopy with (sub)-nanometer axial resolution	32
Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq	32
Integrative structural modeling of macromolecular complexes using Assembline	31
Testing physiologically relevant conditions in minimal inhibitory concentration assays	31
Selected ion flow tube mass spectrometry for targeted analysis of volatile organic compounds in human breath	31
Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues	31
Preparation and quantitative analysis of multicenter luminescence materials for sensing function	31
Subzero non-frozen preservation of human livers in the supercooled state	31
Perfusion and endothelialization of engineered tissues with patterned vascular networks	31
Imaging and optogenetic modulation of vascular mural cells in the live brain	31
Simple, efficient and thorough shotgun proteomic analysis with PatternLab V	31
Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells	30
Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells	30
iBEAT V2.0: a multisite-applicable, deep learning-based pipeline for infant cerebral cortical surface reconstruction	30
Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq	30
Tutorial: structural characterization of isolated metal atoms and subnanometric metal clusters in zeolites	30
Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells	30
Single-crystal structure determination of nanosized metal–organic frameworks by three-dimensional electron diffraction	30
A methodological checklist for fMRI drug cue reactivity studies: development and expert consensus	29
Implementation of a 4Pi-SMS super-resolution microscope	29
Spatial visualization of comprehensive brain neurotransmitter systems and neuroactive substances by selective in situ chemical derivatization mass spectrometry imaging	29
Large-scale site-specific mapping of the O-GalNAc glycoproteome	29
Nuclei isolation of multiple brain cell types for omics interrogation	29
Effective high-throughput isolation of fully human antibodies targeting infectious pathogens	29
Determination of G-protein–coupled receptor oligomerization by molecular brightness analyses in single cells	28
Regional and functional division of functional elements of solid-state nanochannels for enhanced sensitivity and specificity of biosensing in complex matrices	28
Physical dynamic double-network hydrogels as dressings to facilitate tissue repair	28
Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid	28
Preparation of near-infrared AIEgen-active fluorescent probes for mapping amyloid-β plaques in brain tissues and living mice	28
Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy	28
CRISPRi-seq for genome-wide fitness quantification in bacteria	27
A simple sperm-sexing method that activates TLR7/8 on X sperm for the efficient production of sexed mouse or cattle embryos	27
The cell-line-derived subcutaneous tumor model in preclinical cancer research	27
Revealing nascent RNA processing dynamics with nano-COP	27
Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue	27
Bacterial mock communities as standards for reproducible cytometric microbiome analysis	27
Tracing DNA paths and RNA profiles in cultured cells and tissues with ORCA	27
An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli	27
Optimizing the Cell Painting assay for image-based profiling	26
Comprehensive isolation of extracellular vesicles and nanoparticles	26
Recovering prokaryotic genomes from host-associated, short-read shotgun metagenomic sequencing data	26
Hyperosmolar blood–brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance	26
Preparation of robust fluorescent probes for tracking endogenous formaldehyde in living cells and mouse tissue slices	26
Identification, isolation and analysis of human gut-associated lymphoid tissues	26
In vivo compression and imaging in mouse brain to measure the effects of solid stress	26
SPRITE: a genome-wide method for mapping higher-order 3D interactions in the nucleus using combinatorial split-and-pool barcoding	26
Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells	26
Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning	26
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-	26
Directed differentiation of human induced pluripotent stem cells to hepatic stellate cells	26
Transcription factor chromatin profiling genome-wide using uliCUT&RUN in single cells and individual blastocysts	26
Geometric morphometrics of microscopic animals as exemplified by model nematodes	25
Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain	25
Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture	25
Probing low-copy-number proteins in single living cells using single-cell plasmonic immunosandwich assays	25
Precise motor mapping with transcranial magnetic stimulation	25
Genetically modified mouse models to help fight COVID-19	25
Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables	25
Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research	25
Considerations for implementing electronic laboratory notebooks in an academic research environment	24
A graphical user interface to design high-throughput optogenetic experiments with the optoPlate-96	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
Scalable characterization of the PAM requirements of CRISPR–Cas enzymes using HT-PAMDA	24
Rapid in situ identification of biological specimens via DNA amplicon sequencing using miniaturized laboratory equipment	24
Pooled genetic perturbation screens with image-based phenotypes	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
Encapsulating and stabilizing enzymes using hydrogen-bonded organic frameworks	23
Hyperpolarized water as universal sensitivity booster in biomolecular NMR	23
Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria	23
Raman spectral cytopathology for cancer diagnostic applications	23
Isotropic imaging across spatial scales with axially swept light-sheet microscopy	23
Utilizing Skyline to analyze lipidomics data containing liquid chromatography, ion mobility spectrometry and mass spectrometry dimensions	23
Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2)	23
High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening	23
The in vitro multilineage differentiation and maturation of lung and airway cells from human pluripotent stem cell–derived lung progenitors in 3D	23
Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases	23
Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs)	22
A ‘print–pause–print’ protocol for 3D printing microfluidics using multimaterial stereolithography	22
Decoding personal biotic and abiotic airborne exposome	22
Interrogating in vivo T-cell metabolism in mice using stable isotope labeling metabolomics and rapid cell sorting	22
An end-to-end workflow for multiplexed image processing and analysis	22
Quantitative determination of phosphatidylethanol in dried blood spots for monitoring alcohol abstinence	22
Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping	22
Synthesis and characterization of chemically fueled supramolecular materials driven by carbodiimide-based fuels	22
Assessing motor-related phenotypes of Caenorhabditis elegans with the wide field-of-view nematode tracking platform	22