Nature Protocols

Article	Citations
Using MetaboAnalyst 5.0 for LC–HRMS spectra processing, multi-omics integration and covariate adjustment of global metabolomics data	867
The trRosetta server for fast and accurate protein structure prediction	395
CODEX multiplexed tissue imaging with DNA-conjugated antibodies	342
A practical guide to large-scale docking	274
Isolation and characterization of extracellular vesicle subpopulations from tissues	264
Metagenome analysis using the Kraken software suite	236
Proximity labeling in mammalian cells with TurboID and split-TurboID	230
I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction	225
Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays	224
Tutorial: guidelines for the computational analysis of single-cell RNA sequencing data	208
Chromatin accessibility profiling by ATAC-seq	198
MOF-derived nanoporous carbons with diverse tunable nanoarchitectures	186
Artificial intelligence–enabled virtual screening of ultra-large chemical libraries with deep docking	167
Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles	162
Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery	158
Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development	158
Engineering SARS-CoV-2 using a reverse genetic system	153
Tutorial: best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation	149
Measuring DNA modifications with the comet assay: a compendium of protocols	145
Optimization of metabolomic data processing using NOREVA	141
Multiplexed single-cell proteomics using SCoPE2	138
High-yield, green and scalable methods for producing MOF-303 for water harvesting from desert air	138
3D bioprinted silk fibroin hydrogels for tissue engineering	137
High-yield production of mono- or few-layer transition metal dichalcogenide nanosheets by an electrochemical lithium ion intercalation-based exfoliation method	136
Tutorial: guidelines for annotating single-cell transcriptomic maps using automated and manual methods	131

An in vitro batch fermentation protocol for studying the contribution of food to gut microbiota composition and functionality	127
Chemometric analysis in Raman spectroscopy from experimental design to machine learning–based modeling	123
High-throughput cultivation and identification of bacteria from the plant root microbiota	123
Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids	122
Engineered human blood–brain barrier microfluidic model for vascular permeability analyses	119
Intestinal organoid cocultures with microbes	118
Microfluidic integration of regeneratable electrochemical affinity-based biosensors for continual monitoring of organ-on-a-chip devices	113
Algorithmic assessment of cellular senescence in experimental and clinical specimens	113
Generation of insulin-producing pancreatic β cells from multiple human stem cell lines	109
Precision genome editing using cytosine and adenine base editors in mammalian cells	109
A dynamic DNA tetrahedron framework for active targeting	109
Rolling circle amplification (RCA)-based DNA hydrogel	105
Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases	104
RNA-extraction-free nano-amplified colorimetric test for point-of-care clinical diagnosis of COVID-19	101
Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing	99
Microtiter plate assays to assess antibiofilm activity against bacteria	98
Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing	97
3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert	94
Analyzing high-dimensional cytometry data using FlowSOM	93
A metabolic modeling platform for the computation of microbial ecosystems in time and space (COMETS)	92
The normative modeling framework for computational psychiatry	90
Physical dynamic double-network hydrogels as dressings to facilitate tissue repair	87
Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging	87
Establishment of human fetal hepatocyte organoids and CRISPR–Cas9-based gene knockin and knockout in organoid cultures from human liver	87
Generic acquisition protocol for quantitative MRI of the spinal cord	78
Engineering brain assembloids to interrogate human neural circuits	77
Comprehensive isolation of extracellular vesicles and nanoparticles	75
Detection of aberrant gene expression events in RNA sequencing data	75
Synthesis of siRNA nanoparticles to silence plaque-destabilizing gene in atherosclerotic lesional macrophages	73
Genetic circuit design automation with Cello 2.0	72
Measuring the elastic modulus of soft culture surfaces and three-dimensional hydrogels using atomic force microscopy	72
Fabrication, characterization and applications of graphene electronic tattoos	71
Tools for experimental and computational analyses of off-target editing by programmable nucleases	70
A robust unsupervised machine-learning method to quantify the morphological heterogeneity of cells and nuclei	70
Improved high-molecular-weight DNA extraction, nanopore sequencing and metagenomic assembly from the human gut microbiome	69
Nanocarriers based on bacterial membrane materials for cancer vaccine delivery	69
NovoSpaRc: flexible spatial reconstruction of single-cell gene expression with optimal transport	69
Digital postprocessing and image segmentation for objective analysis of colorimetric reactions	68
Transplantation of intestinal organoids into a mouse model of colitis	67
Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH	67
Whole-brain functional ultrasound imaging in awake head-fixed mice	67
Accurate determination of protein:ligand standard binding free energies from molecular dynamics simulations	67
IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues	66
Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid	64
Raman spectroscopy for real-time and in situ monitoring of mechanochemical milling reactions	64
Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity	63
Cargo loading within ferritin nanocages in preparation for tumor-targeted delivery	63
A well plate–based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature	62
Preparation and quantitative analysis of multicenter luminescence materials for sensing function	61
Tutorial: practical considerations for tissue clearing and imaging	61

Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells	60
Tutorial: using nanoneedles for intracellular delivery	60
Stem-cell-derived human microglia transplanted into mouse brain to study human disease	60
Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq	59
Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues	57
Designing and executing prime editing experiments in mammalian cells	57
Monomicellar assembly to synthesize structured and functional mesoporous carbonaceous nanomaterials	57
Analysis of clonogenic growth in vitro	57
Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope	56
Stable isotope tracing to assess tumor metabolism in vivo	56
Design and simulation of DNA, RNA and hybrid protein–nucleic acid nanostructures with oxView	56
Synthesis of N-heterocyclic carbene gold(I) complexes	55
Evaluation of the effects of cold atmospheric plasma and plasma-treated liquids in cancer cell cultures	54
Culture and analysis of kidney tubuloids and perfused tubuloid cells-on-a-chip	54
Phage-assisted continuous and non-continuous evolution	54
Optimized single-nucleus transcriptional profiling by combinatorial indexing	53
Characterization of connective tissues using near-infrared spectroscopy and imaging	53
Use of deep learning to develop continuous-risk models for adverse event prediction from electronic health records	51
Mapping mechanical properties of biological materials via an add-on Brillouin module to confocal microscopes	51
An end-to-end workflow for multiplexed image processing and analysis	50
Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells	50
Preparation of near-infrared AIEgen-active fluorescent probes for mapping amyloid-β plaques in brain tissues and living mice	50
Inducible mouse models of colon cancer for the analysis of sporadic and inflammation-driven tumor progression and lymph node metastasis	49
Design, assembly, and characterization of membrane-spanning DNA nanopores	49
Implantation of Neuropixels probes for chronic recording of neuronal activity in freely behaving mice and rats	49
Optimizing the Cell Painting assay for image-based profiling	48
Statistical analysis in metabolic phenotyping	47
Fabrication of liquid cell for in situ transmission electron microscopy of electrochemical processes	47
In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels	47
Creating hierarchical pores in metal–organic frameworks via postsynthetic reactions	47
Continuous human iPSC-macrophage mass production by suspension culture in stirred tank bioreactors	46
High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity	46
iBEAT V2.0: a multisite-applicable, deep learning-based pipeline for infant cerebral cortical surface reconstruction	45
Chronic, cortex-wide imaging of specific cell populations during behavior	45
CRISPRi-seq for genome-wide fitness quantification in bacteria	45
Spooling electrochemiluminescence spectroscopy: development, applications and beyond	44
Perfusion and endothelialization of engineered tissues with patterned vascular networks	44
Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions	44
Single-crystal structure determination of nanosized metal–organic frameworks by three-dimensional electron diffraction	43
All-optical interrogation of neural circuits in behaving mice	42
Expression and characterization of SARS-CoV-2 spike proteins	42
Composable microfluidic spinning platforms for facile production of biomimetic perfusable hydrogel microtubes	42
Testing physiologically relevant conditions in minimal inhibitory concentration assays	41
A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility	41
Graphene–molecule–graphene single-molecule junctions to detect electronic reactions at the molecular scale	40
A methodological checklist for fMRI drug cue reactivity studies: development and expert consensus	40
Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues	39
Quantification of organelle contact sites by split-GFP-based contact site sensors (SPLICS) in living cells	39
Simple, efficient and thorough shotgun proteomic analysis with PatternLab V	39
Smart-RRBS for single-cell methylome and transcriptome analysis	39
Graphene- and metal-induced energy transfer for single-molecule imaging and live-cell nanoscopy with (sub)-nanometer axial resolution	38
Protein higher-order-structure determination by fast photochemical oxidation of proteins and mass spectrometry analysis	38
Coculture of primary human colon monolayer with human gut bacteria	38
Selected ion flow tube mass spectrometry for targeted analysis of volatile organic compounds in human breath	38
Nuclei isolation of multiple brain cell types for omics interrogation	38
Tutorial: assessing metagenomics software with the CAMI benchmarking toolkit	38
Uncovering structural ensembles from single-particle cryo-EM data using cryoDRGN	38
Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq	38
Regional and functional division of functional elements of solid-state nanochannels for enhanced sensitivity and specificity of biosensing in complex matrices	37
Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq	37
An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity	37
Integrative structural modeling of macromolecular complexes using Assembline	37
Proteome-wide structural changes measured with limited proteolysis-mass spectrometry: an advanced protocol for high-throughput applications	37
Responsive and activable nanomedicines for remodeling the tumor microenvironment	36
Spatial visualization of comprehensive brain neurotransmitter systems and neuroactive substances by selective in situ chemical derivatization mass spectrometry imaging	36
Effective high-throughput isolation of fully human antibodies targeting infectious pathogens	36
The maternal serum metabolome by multisegment injection-capillary electrophoresis-mass spectrometry: a high-throughput platform and standardized data workflow for large-scale epidemiological studies	36
High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery	35
The cell-line-derived subcutaneous tumor model in preclinical cancer research	35
Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells	34
QurvE: user-friendly software for the analysis of biological growth and fluorescence data	34
Encapsulating and stabilizing enzymes using hydrogen-bonded organic frameworks	34
Imaging and optogenetic modulation of vascular mural cells in the live brain	34
Implementation of a 4Pi-SMS super-resolution microscope	34
Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture	34
Hyperosmolar blood–brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance	34
Direct delivery and fast-treated Agrobacterium co-culture (Fast-TrACC) plant transformation methods for Nicotiana benthamiana	33
Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells	33
Tracing DNA paths and RNA profiles in cultured cells and tissues with ORCA	33
Utilizing Skyline to analyze lipidomics data containing liquid chromatography, ion mobility spectrometry and mass spectrometry dimensions	33

Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research	33
Considerations for implementing electronic laboratory notebooks in an academic research environment	33
Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures	33
Precise motor mapping with transcranial magnetic stimulation	33
Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy	32
SPRITE: a genome-wide method for mapping higher-order 3D interactions in the nucleus using combinatorial split-and-pool barcoding	32
Acoustic tweezers for high-throughput single-cell analysis	32
High-throughput proteomic sample preparation using pressure cycling technology	32
Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning	31
Scale-up approach for the preparation of magnetic ferrite nanocubes and other shapes with benchmark performance for magnetic hyperthermia applications	31
Recovering prokaryotic genomes from host-associated, short-read shotgun metagenomic sequencing data	31
Hyperpolarized water as universal sensitivity booster in biomolecular NMR	30
A ‘print–pause–print’ protocol for 3D printing microfluidics using multimaterial stereolithography	30
A checklist for assessing the methodological quality of concurrent tES-fMRI studies (ContES checklist): a consensus study and statement	30
Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture	30
Directed differentiation of human induced pluripotent stem cells to hepatic stellate cells	30
Isotropic imaging across spatial scales with axially swept light-sheet microscopy	30
Interrogating in vivo T-cell metabolism in mice using stable isotope labeling metabolomics and rapid cell sorting	30
Revealing nascent RNA processing dynamics with nano-COP	30
Identification, isolation and analysis of human gut-associated lymphoid tissues	29
Transcription factor chromatin profiling genome-wide using uliCUT&RUN in single cells and individual blastocysts	29
Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain	29
Click chemistry: a transformative technology in nuclear medicine	29
An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli	29
Enzyme immobilization on covalent organic framework supports	29
Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2)	28
Rapid in situ identification of biological specimens via DNA amplicon sequencing using miniaturized laboratory equipment	28
Determination of G-protein–coupled receptor oligomerization by molecular brightness analyses in single cells	28
Genome-wide detection of DNA double-strand breaks by in-suspension BLISS	28
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-	28
Raman spectral cytopathology for cancer diagnostic applications	28
Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria	28
A practical guide to machine-learning scoring for structure-based virtual screening	28
Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue	28
Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs)	27
rMATS-turbo: an efficient and flexible computational tool for alternative splicing analysis of large-scale RNA-seq data	27
Nacre-inspired underwater superoleophobic films with high transparency and mechanical robustness	27
Pooled genetic perturbation screens with image-based phenotypes	27
Multiplexed single-cell analysis of organoid signaling networks	27
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	26
The in vitro multilineage differentiation and maturation of lung and airway cells from human pluripotent stem cell–derived lung progenitors in 3D	26
Scalable tissue labeling and clearing of intact human organs	26
Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables	26
Whole-mouse clearing and imaging at the cellular level with vDISCO	26
Probing low-copy-number proteins in single living cells using single-cell plasmonic immunosandwich assays	26
Comprehensive synthesis of Ti3C2Tx from MAX phase to MXene	26
Generation of heart-forming organoids from human pluripotent stem cells	26
Prioritization of cell types responsive to biological perturbations in single-cell data with Augur	26
Inverse electron demand Diels–Alder click chemistry for pretargeted PET imaging and radioimmunotherapy	26
Using clusterProfiler to characterize multiomics data	26
Exposure protocol for ecotoxicity testing of microplastics and nanoplastics	25
Global in situ profiling of RNA-RNA spatial interactions with RIC-seq	25
Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases	25
Reaction screening in multiwell plates: high-throughput optimization of a Buchwald–Hartwig amination	25
Scalable characterization of the PAM requirements of CRISPR–Cas enzymes using HT-PAMDA	25
Scaling up reproducible research for single-cell transcriptomics using MetaNeighbor	25
In situ electrochemical Raman spectroscopy and ab initio molecular dynamics study of interfacial water on a single-crystal surface	25
Solution-state nuclear magnetic resonance spectroscopy of crystalline cellulosic materials using a direct dissolution ionic liquid electrolyte	24
Mitochondrial single-cell ATAC-seq for high-throughput multi-omic detection of mitochondrial genotypes and chromatin accessibility	24
Generation of neighbor-labeling cells to study intercellular interactions in vivo	24
Nondestructive production of exosomes loaded with ultrathin palladium nanosheets for targeted bio-orthogonal catalysis	24
Infection of zebrafish larvae with human norovirus and evaluation of the in vivo efficacy of small-molecule inhibitors	24
OCTAD: an open workspace for virtually screening therapeutics targeting precise cancer patient groups using gene expression features	24
Constructing a cost-efficient, high-throughput and high-quality single-molecule localization microscope for super-resolution imaging	24
Decoding personal biotic and abiotic airborne exposome	24
Measuring key human carbohydrate digestive enzyme activities using high-performance anion-exchange chromatography with pulsed amperometric detection	23
Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping	23
Spatial- and Fourier-domain ptychography for high-throughput bio-imaging	23
Web-based multi-omics integration using the Analyst software suite	23
The HADDOCK2.4 web server for integrative modeling of biomolecular complexes	23
Metagenome-assembled genome extraction and analysis from microbiomes using KBase	23
Deepometry, a framework for applying supervised and weakly supervised deep learning to imaging cytometry	23
Synthesis and characterization of chemically fueled supramolecular materials driven by carbodiimide-based fuels	23
KAS-seq: genome-wide sequencing of single-stranded DNA by N3-kethoxal–assisted labeling	22
m6A-SAC-seq for quantitative whole transcriptome m6A profiling	22
Fabricating and printing chemiresistors based on monolayer-capped metal nanoparticles	22
Quantitative determination of phosphatidylethanol in dried blood spots for monitoring alcohol abstinence	22
Quantum defects as versatile anchors for carbon nanotube functionalization	22
Near-infrared catecholamine nanosensors for high spatiotemporal dopamine imaging	22
CRISPR–Cas9-mediated chromosome engineering in Arabidopsis thaliana	22
Detecting chromosomal interactions in Capture Hi-C data with CHiCAGO and companion tools	21
Monitoring mammalian mitochondrial translation with MitoRiboSeq	21
Making bioinspired 3D-printed autonomic perspiring hydrogel actuators	21
Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing	21
Protein transfection via spherical nucleic acids	21
Fabrication and use of silicon hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo	20
Combining surface-accessible Ag and Au colloidal nanomaterials with SERS for in situ analysis of molecule–metal interactions in complex solution environments	20
Identifying genome-wide off-target sites of CRISPR RNA–guided nucleases and deaminases with Digenome-seq	20
Lineage barcoding in mice with homing CRISPR	20
Preparation and use of wireless reprogrammable multilateral optogenetic devices for behavioral neuroscience	20
Metabolic labeling of secreted matrix to investigate cell–material interactions in tissue engineering and mechanobiology	20
Development of ionic liquid-coated PLGA nanoparticles for applications in intravenous drug delivery	20
Genetic in vivo engineering of human T lymphocytes in mouse models	20
Quantitative magnetic resonance imaging and tumor forecasting of breast cancer patients in the community setting	20
Interrogation of the mammalian gut–brain axis using LC–MS/MS-based targeted metabolomics with in vitro bacterial and organoid cultures and in vivo gnotobiotic mouse models	20