High-throughput Isolation Research Articles

A novel high-throughput single B-cell cloning platform for isolation and characterization of high-affinity and potent SARS-CoV-2 neutralizing antibodies

Monoclonal antibodies (mAbs) that are specific to SARS-CoV-2 can be useful in diagnosing, preventing, and treating the coronavirus (COVID-19) illness. Strategies for the high-throughput and rapid isolation of these potent neutralizing antibodies are critical toward the development of therapeutically targeting COVID-19 as well as other infectious diseases. In the present study, a single B-cell cloning method was used to screen the Wuhan-Hu-1 strain of SARS-CoV-2 receptor-binding domain (RBD) specific, high affinity, and neutralizing mAbs from patients’ blood samples. An RBD-specific antibody, SAR03, was discovered that showed high binding (ELISA and SPR) and neutralizing activity (competitive ELISA and pseudovirus-based reporter assay) against the Wuhan-Hu-1 strain of SARS-CoV-2. Mechanistic studies on human cells revealed that SAR03 competes with the ACE-2 receptor for binding with the RBD domain (S1 subunit) present in the spike protein of SARS-CoV-2. This study highlights the potential of the single B cell cloning method for the rapid and efficient screening of high-affinity and effective neutralizing antibodies for SARS-CoV-2 and other emerging infectious diseases.

Microprobe‐based technology for antigen‐specific purification of exosomes for OMICS analysis

Exosomes encapsulate genomic and proteomic biomarkers for non‐invasive diagnosis and disease monitoring. However, exosome surface‐markers heterogeneity is a major drawback of current isolation methods. Here, we report a direct, one‐step exosome sampling technology, ExoPRIME, for selective capture of CD63+ exosome subpopulations using an immune‐affinity protocol. Microneedles (300μm × 30 mm), functionalized with anti‐ CD63 antibodies, were incubated under various experimental conditions in conditioned astrocyte medium and astrocyte‐derived exosome suspension. The probe’s capture efficiency and specificity were validated using Flu‐oroCet assay, immunofluorescent imaging, and OMICS analyses. Significantly higher exosomes were captured by probes incubated for 16 h at 4 0C in enriched exosomal suspension (23 × 10 6 exosomes per probe) vis‐'a‐vis 2 h at 4 0 C (12 × 10 6) and 16 h at 22 0C (3 × 10 6) in conditioned cell media. Our results demonstrate the application of ExoPRIME over a broad dynamic range of temperature and incubation parameters, offering flexibility for any desired application. ExoPRIME permits the use and re‐use of minimal sample volumes (≤200 μL), can be multiplexed in arrays, and integrated into a lab‐on‐a‐chip platform to achieve parallel, high‐throughput isolation of different exosome classes in a semi‐automated workstation. This platform could provide direct exosomal analysis of biological fluids since it can elegantly interface with existing room‐temperature, picomolar‐range nucleic acid assays to provide a clinical diagnostic tool at the point of care.

Effective high-throughput isolation of enriched platelets and circulating pro-angiogenic cells to accelerate skin-wound healing

Delayed wound healing and chronic skin lesions represent a major health problem. Over the past years, growth factors mediated by platelet-rich plasma (PRP) and cell-based therapies were developed as effective and affordable treatment able to improve wound healing capacity. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of PRP and pro-angiogenic cells (AngioPRP). This protocol outlines the effectiveness of AngioPRP in promoting the critical healing process including wound closure, re-epithelialization, granulation tissue growth, and blood vessel regeneration. We coupled this effect with normalization of mechanical properties of rescued mouse wounds, which is sustained by a correct arrangement of elastin and collagen fibers. Proteomic analysis of treated wounds demonstrated a fingerprint of AngioPRP based on the up-regulation of detoxification pathway of glutathione metabolism, correlated to a decrease in inflammatory response. Overall, these results have enabled us to provide a framework for how AngioPRP supports wound healing, opening avenues for further clinical advances.

Extended characterisation of five archival tick-borne viruses provides insights for virus discovery in Australian ticks

BackgroundA subset of Australians who have been bitten by ticks experience a complex of chronic and debilitating symptoms which cannot be attributed to the known pathogenic species of bacteria present in Australia. As a result, there has been a renewed effort to identify and characterise viruses in Australian terrestrial ticks. Recent transcriptome sequencing of Ixodes and Amblyomma ticks has revealed the presence of multiple virus sequences. However, without virus isolates our ability to understand the host range and pathogenesis of newly identified viruses is limited. We have established a successful method for high-throughput virus discovery and isolation in mosquitoes using antibodies to double-stranded RNA. In this study we sought to characterise five archival tick-borne viruses to adapt our virus discovery protocol for Australian ticks.MethodsWe performed virus characterisation using a combination of bioinformatic sequence analysis and in vitro techniques including replication kinetics, antigenic profiling, virus purification and mass spectrometry.ResultsOur sequence analysis of Nugget virus, Catch-me-Cave virus and Finch Creek virus revealed marked genetic stability in isolates collected from the same location approximately 30 years apart. We demonstrate that the Ixodes scapularis-derived ISE6 cell line supports replication of Australian members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families, including Saumarez Reef virus (SREV), a flavivirus isolated from the soft tick Ornithodoros capensis. While antibodies against double-stranded RNA could be used to detect replication of a tick-borne reovirus and mosquito-borne flavivirus, the tick-borne flaviviruses Gadgets Gully virus and SREV could not be detected using this method. Finally, four novel virus-like sequences were identified in transcriptome sequencing of the Australian native tick Ixodes holocyclus.ConclusionsGenetic and antigenic characterisations of archival viruses in this study confirm that three viruses described in 2002 represent contemporary isolates of virus species first identified 30 years prior. Our findings with antibodies to double-stranded RNA highlight an unusual characteristic shared by two Australian tick-borne flaviviruses. Finally, comparative growth kinetics analyses of Australian tick-borne members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families in ISE6 and BSR cells will provide a useful resource for isolation of Australian tick-borne viruses using existing cell lines.Graphical

Integrated microfluidic single-cell immunoblotting chip enables high-throughput isolation, enrichment and direct protein analysis of circulating tumor cells

Effective capture and analysis of a single circulating tumor cell (CTC) is instrumental for early diagnosis and personalized therapy of tumors. However, due to their extremely low abundance and susceptibility to interference from other cells, high-throughput isolation, enrichment, and single-cell-level functional protein analysis of CTCs within one integrated system remains a major challenge. Herein, we present an integrated multifunctional microfluidic system for highly efficient and label-free CTC isolation, CTC enrichment, and single-cell immunoblotting (ieSCI). The ieSCI-chip is a multilayer microfluidic system that combines an inertia force-based cell sorter with a membrane filter for label-free CTC separation and enrichment and a thin layer of a photoactive polyacrylamide gel with microwell arrays at the bottom of the chamber for single-cell immunoblotting. The ieSCI-chip successfully identified a subgroup of apoptosis-negative (Bax-negative) cells, which traditional bulk analysis did not detect, from cisplatin-treated cells. Furthermore, we demonstrated the clinical application of the ieSCI-chip with blood samples from breast cancer patients for personalized CTC epithelial-to-mesenchymal transition (EMT) analysis. The expression level of a tumor cell marker (EpCAM) can be directly determined in isolated CTCs at the single-cell level, and the therapeutic response to anticancer drugs can be simultaneously monitored. Therefore, the ieSCI-chip provides a promising clinical translational tool for clinical drug response monitoring and personalized regimen development.

Rapid and Highly Efficient Isolation and Purification of Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) hold great promise for biomedical applications. However, establishment of new iPSC lines still presents many challenges. Here we describe a simple yet highly efficient two-step protocol for the isolation and purification of human iPSC lines. The first step adapts iPSCs to single cell culture and passaging, promoting survival and self-renewal; the second step enables the isolation and purification of bona fide iPSCs from a mixed population using column-based positive selection of cells expressing pluripotency markers such as TRA-1-60. Both steps utilize commercially available reagents. Using this protocol, iPSCs can be purified from cell preparations containing differentiated or unreprogrammed cells, or even be isolated directly from reprogramming vessels. The protocol could be adopted for high throughput isolation and expansion of iPSC lines and facilitate the widespread use of iPSCs in future applications.

ExoPRIME: Solid-phase immunoisolation and OMICS analysis of surface-marker-specific exosomal subpopulations

Exosomes encapsulate genomic and proteomic biomarkers for non-invasive diagnosis and disease monitoring. However, exosome surface-markers heterogeneity is a major drawback of current isolation methods. Here, we report a direct, one-step exosome sampling technology, ExoPRIME, for selective capture of CD63+ exosome subpopulations using an immune-affinity protocol. Microneedles (300μm × 30 mm), functionalized with anti-CD63 antibodies, were incubated under various experimental conditions in conditioned astrocyte medium and astrocyte-derived exosome suspension. The probe's capture efficiency and specificity were validated using FluoroCet assay, immunofluorescent imaging, and OMICS analyses. Significantly higher exosomes were captured by probes incubated for 16 h at 4 0C in enriched exosomal suspension (23 × 10 6 exosomes per probe) vis-à-vis 2 h at 4 0 C (12 × 10 6) and 16 h at 22 0C (3 × 10 6) in conditioned cell media. Our results demonstrate the application of ExoPRIME over a broad dynamic range of temperature and incubation parameters, offering flexibility for any desired application. ExoPRIME permits the use and re-use of minimal sample volumes (≤200 μL), can be multiplexed in arrays, and integrated into a lab-on-a-chip platform to achieve parallel, high-throughput isolation of different exosome classes in a semi-automated workstation. This platform could provide direct exosomal analysis of biological fluids since it can elegantly interface with existing room-temperature, picomolar-range nucleic acid assays to provide a clinical diagnostic tool at the point of care.

Assessing the Cultivability of Bacteria and Fungi from Arable Crop Residues Using Metabarcoding Data as a Reference

This study combined culture-dependent (strain isolation plus molecular identification) and culture-independent (metabarcoding) approaches to characterize the diversity of microbiota on wheat and oilseed rape residues. The goal was to develop a methodology to culture microorganisms with the aim of being able to establish synthetic crop residue microbial communities for further study, i.e., testing potential interactions within these communities and characterizing groups of beneficial taxa that could be used as biological control agents against plant pathogens. We generated community-based culture collections. We adapted the isolation strategy to the potential differences in the spatial and temporal distribution of diversity between bacteria and fungi. We performed (i) a high-throughput isolation from few samples with no a priori for bacteria and (ii) a low-throughput isolation from several samples with a priori—i.e., morphotype selection—for fungi. Although isolation using a single medium did not allow us to characterize the microbiome as precisely as metabarcoding, the bacterial diversity (158 ASVs, 36 genera) was relatively higher than the fungal diversity (131 ASVs, 17 genera) known to be limited by competition for growth on non-selective solid media. Isolation and metabarcoding provided consistent and complementary information: they revealed several common but also specific ASVs, leading to close microbial community profiles of the most abundant fungal and bacterial taxa in residues. Finally, by empirically comparing the different profiles, we assessed the cultivability of the most abundant fungal and bacterial taxa obtained in metabarcoding.

Clog-free high-throughput microfluidic cell isolation with multifunctional microposts

Microfluidics have been applied to filtration of rare tumor cells from the blood as liquid biopsies. Processing is highly limited by low flow rates and device clogging due to a single function of fluidic paths. A novel method using multifunctional hybrid functional microposts was developed. A swift by-passing route for non-tumor cells was integrated to prevent very common clogging problems. Performance was characterized using microbeads (10 µm) and human cancer cells that were spiked in human blood. Design-I showed a capture efficiency of 96% for microbeads and 87% for cancer cells at 1 ml/min flow rate. An improved Design-II presented a higher capture efficiency of 100% for microbeads and 96% for cancer cells. Our method of utilizing various microfluidic functions of separation, bypass and capture has successfully guaranteed highly efficient separation of rare cells from biological fluids.

Antimicrobial Peptides from Plants: A cDNA-Library Based Isolation, Purification, Characterization Approach and Elucidating Their Modes of Action.

Even in a natural ecosystem, plants are continuously threatened by various microbial diseases. To save themselves from these diverse infections, plants build a robust, multilayered immune system through their natural chemical compounds. Among the several crucial bioactive compounds possessed by plants’ immune systems, antimicrobial peptides (AMPs) rank in the first tier. These AMPs are environmentally friendly, anti-pathogenic, and do not bring harm to humans. Antimicrobial peptides can be isolated in several ways, but recombinant protein production has become increasingly popular in recent years, with the Escherichia coli expression system being the most widely used. However, the efficacy of this expression system is compromised due to the difficulty of removing endotoxin from its system. Therefore, this review suggests a high-throughput cDNA library-based plant-derived AMP isolation technique using the Bacillus subtilis expression system. This method can be performed for large-scale screening of plant sources to classify unique or homologous AMPs for the agronomic and applied field of plant studies. Furthermore, this review also focuses on the efficacy of plant AMPs, which are dependent on their numerous modes of action and exceptional structural stability to function against a wide range of invaders. To conclude, the findings from this study will be useful in investigating how novel AMPs are distributed among plants and provide detailed guidelines for an effective screening strategy of AMPs.

Dielectric Characterization and Multistage Separation of Various Cells via Dielectrophoresis in a Bipolar Electrode Arrayed Device.

Isolation of microalgal cells is as an indispensable part of producing biofuels for energy security and detecting toxic contaminants for marine routine monitoring. Microalgae live together with various microalgae naturally, and abundant samples need to be tackled in practical applications. Therefore, effective separation technologies need to be developed urgently to achieve high-throughput separation of various microalgae. Herein, we develop a reliable device to characterize the dielectric response of microalgae and sequentially separate various microalgae utilizing dielectrophoretic force in a bipolar electrode (BPE) arrayed device. First, by investigating the array width extension (AWE) effect on the electric- and flow-field distributions, we explore consequences of incidental electrohydrodynamic mechanisms and axial flow rate on the separation. Second, based on device performance on sample characterizations, we demonstrate this technology by separating microparticles in three- and five-channel devices. Third, we discriminate dead and live cells to explore its capability using the cell viability test and illustrate the AWE influence on the separation. Fourth, we characterize dielectric responses of different microalgae and separate C. vulgaris and Oocystis sp. Finally, we extended BPEs in length and developed an arrayed device for sequential separation of various microalgae, and this platform is successfully engineered in high-throughput isolation of C. vulgaris from complex samples. This technology presents good potential in addressing depleting fossil fuel and burgeoning environmental concerns due to its performance in the separation of microalgal strains from complex samples.

(Nanocarbons Division Robert C. Haddon Research Award) Chemical Functionalization of Synthetic Carbon Allotropes

Chemical functionalization of new C-allotropes is of fundamental interest and opens the door to unprecedented materials applications. In principle, the physical and chemical properties of fullerenes, carbon nanotubes (CNTs) and graphene are related to each other, although their levels of development vary considerably. In order to efficiently explore the reactivity of the less developed CNTs and especially that of graphene it is our goal to provide a unifying approach for the chemistry of all three new carbon allotropes. The fullerenes present the first family of synthetic carbon allotropes. Since their availability in macroscopic quantities numerous investigations with respect to their physical characterization and chemical functionalization have been carried out. CNTs - the second new family of C-allotropes - exhibit at least the same potential for unprecedented applications. Their chemistry, however, is much less developed. Although many protocols for covalent and non-covalent CNT functionalization have been published there are still many fundamental problems to be solved. This includes inter alia the highly selective functionalization of metallic- or semiconducting SWNTs, the high throughput isolation of SWNTs with single helicity, the development of general concepts allowing for tunable doping of individualized tubes with single helicity or least defined transport characteristics (metallic – semiconductive). Finally, the youngest representative in the list of new C-allotropes is graphene and its chemical functionalization opens exciting opportunities towards the development of precisely structured high performance materials. We will talk on the systematic investigation of the chemical properties of graphene both in homogenous solution and deposited on solid substrates. We have revealed fundamental reactivity principles, such as role of defects, charging and substrate. Moreover, we have disclosed for the first time basic fundamental topicity concepts. This allows us to tailor highly integrated graphene based 2D-materials. An example is the introduction of reversible covalent 2D-structuring providing the basis for chemical data storage.

A low‐cost and high‐throughput benchtop cell sorter for isolating white blood cells from whole blood

The ability to isolate and purify white blood cells (WBCs) from mixed ensembles such as blood would benefit autologous cell-based therapeutics as well as diagnosis of WBC disorders. Current WBCs isolation methods have the limitations of low purity or requiring complex and expensive equipment. In addition, due to the overlap in size distribution between lymphocytes (i.e., a sub-population of WBCs) and red blood cells (RBCs), it is challenging to achieve isolation of entire WBCs populations. In this work, we developed an inertial microfluidics-based cell sorter, which enables size-based, high-throughput isolation, and enrichment of WBCs from RBC-lysed whole blood. Using the developed inertial microfluidic chip, the sorting resolution is sharpened within 2μm, which achieved separation between 3 and 5μm diameter particles. Thus, with the present cell sorter, a full population of WBCs can be isolated from RBC-lysed blood samples with recovery ratio of 92%, and merely 5% difference in the composition percentage of the three subpopulations of granulocytes, monocytes, and lymphocytes compared to the original sample. Furthermore, our cell sorter is designed to enable broad application of size-based inertial cell sorting by supplying a series of microchips with different sorting cutoff size. This strategy allows us to further enrich the lymphocytes population by twofold using another microchip with a cutoff size between 10 and 15μm. With simplicity and efficiency, our cell sorter provides a powerful platform for isolating and sorting of WBCs and also envisions broad potential sorting applications for other cell types.

Effective high-throughput isolation of fully human antibodies targeting infectious pathogens.

As exemplified by the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there is a strong demand for rapid high-throughput isolation pipelines to identify potent neutralizing antibodies for prevention and therapy of infectious diseases. However, despite substantial progress and extensive efforts, the identification and production of antigen-specific antibodies remains labor- and cost-intensive. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of potent antigen-specific antibodies against human immunodeficiency virus 1, hepatitis C virus, human cytomegalovirus, Middle East respiratory syndrome coronavirus, SARS-CoV-2 and Ebola virus. It is based on computationally optimized multiplex primer sets (openPrimeR), which guarantee high coverage of even highly mutated immunoglobulin gene segments as well as on optimized antibody cloning and production strategies. Here, we provide the detailed protocol, which covers all critical steps from sample collection to antibody production within 12-14 d.

High-throughput enrichment and isolation of megakaryocyte progenitor cells from the mouse bone marrow

Megakaryocytes are a rare population of cells that develop in the bone marrow and function to produce platelets that circulate throughout the body and form clots to stop or prevent bleeding. A major challenge in studying megakaryocyte development, and the diseases that arise from their dysfunction, is the identification, classification, and enrichment of megakaryocyte progenitor cells that are produced during hematopoiesis. Here, we present a high throughput strategy for identifying and isolating megakaryocytes and their progenitor cells from a heterogeneous population of bone marrow samples. Specifically, we couple thrombopoietin (TPO) induction, image flow cytometry, and principal component analysis (PCA) to identify and enrich for megakaryocyte progenitor cells that are capable of self-renewal and directly differentiating into mature megakaryocytes. This enrichment strategy distinguishes megakaryocyte progenitors from other lineage-committed cells in a high throughput manner. Furthermore, by using image flow cytometry with PCA, we have identified a combination of markers and characteristics that can be used to isolate megakaryocyte progenitor cells using standard flow cytometry methods. Altogether, these techniques enable the high throughput enrichment and isolation of cells in the megakaryocyte lineage and have the potential to enable rapid disease identification and diagnoses ahead of severe disease progression.

Cyclic tangential flow filtration system for isolation of extracellular vesicles.

Size-based filtration techniques have been developed for high-throughput isolation of extracellular vesicles (EVs). Conventional direct filtration systems have limitations in that large particles generally not only block the pores of the membrane but also damage the particles because of the high fluid pressure. Here, we propose a cyclic tangential flow filtration (TFF) system that includes two membranes with pore sizes of 200 and 30 nm, connected to a peristaltic pump that feeds the stream flowing to the membrane for continuous circulation. The cyclic TFF system is better able to isolate the specific 30–200 nm size range in one step through dual cyclic filtration compared with direct filtration (DF) and single cyclic TFF (scTFF). We further introduced a buffer-exchange process to the dcTFF system after filtration to remove contaminants for more efficient purification. As a result of comparative evaluation of dcTFF and ExoQuick, EVs isolated by dcTFF had more abundant exosome markers and active EVs. The cyclic TFF system not only has great potential to separate EVs with high selectivity and separation efficiency in small volumes of samples but can also be used in clinical applications, including medical diagnostic procedures.

Landscape of Monoclonal Antibodies Targeting Zika and Dengue: Therapeutic Solutions and Critical Insights for Vaccine Development.

The unprecedented 2015–2016 Zika outbreak in the Americas sparked global concern and drove the rapid deployment of vaccine and therapeutic countermeasures against this re-emerging pathogen. Alongside vaccine development, a number of potent neutralizing antibodies against Zika and related flaviviruses have been identified in recent years. High-throughput antibody isolation approaches have contributed to a better understanding of the B cell responses elicited following infection and/or vaccination. Structure-based approaches have illuminated species-specific and cross-protective epitopes of therapeutic value. This review will highlight previously described monoclonal antibodies with the best therapeutic potential against ZIKV and related flaviviruses, and discuss their implications for the rational design of better vaccine strategies.

Single-step equipment-free extracellular vesicle concentration using super absorbent polymer beads.

Extracellular vesicles (EVs) contain useful biomarkers for disease diagnosis and are promising biomaterials for the delivery of therapeutic molecules in vivo. Accordingly, an efficient concentration method is necessary for large‐scale production or high‐throughput isolation of EVs from bulk liquid samples, including culture medium and body fluids, to achieve their clinical application. However, current EV concentration methods, including ultrafiltration, are limited with respect to cost, efficiency, and centrifugation time. In this study, we developed the first single‐step, equipment‐free EV concentration method using super absorbent polymer (SAP) beads. SAP beads absorb small molecules, including water, via nano‐sized channels but expel and thereby concentrate EVs. Consequently, the beads drastically enrich EVs by reducing the solution volume in a single step, without affecting EV characteristics. Moreover, the purity of the concentrated EV solution was high due to the absorption of protein impurities by SAP beads. To further demonstrate the versatility of the method, we showed that SAP beads successfully enrich EVs in human urine samples and culture medium, enabling better isolation performance than conventional ultrafiltration. We believe the newly developed approach and insight gained in this study will facilitate the use of EVs as prominent biomaterials for disease diagnosis and therapy.

The Microfluidic Toolbox for Analyzing Exosome Biomarkers of Aging.

As the fields of aging and neurological disease expand to liquid biopsies, there is a need to identify informative biomarkers for the diagnosis of neurodegeneration and other age-related disorders such as cancers. A means of high-throughput screening of biomolecules relevant to aging can facilitate this discovery in complex biofluids, such as blood. Exosomes, the smallest of extracellular vesicles, are found in many biofluids and, in recent years, have been found to be excellent candidates as liquid biopsy biomarkers due to their participation in intercellular communication and various pathologies such as cancer metastasis. Recently, exosomes have emerged as novel biomarkers for age-related diseases. Hence, the study of exosomes, their protein and genetic cargo can serve as early biomarkers for age-associated pathologies, especially neurodegenerative diseases. However, a disadvantage of exosome studies includes a lack in standardization of isolating, detecting, and profiling exosomes for downstream analysis. In this review, we will address current techniques for high-throughput isolation and detection of exosomes through various microfluidic and biosensing strategies and how they may be adapted for the detection of biomarkers of age-associated disorders.

High-throughput cultivation and identification of bacteria from the plant root microbiota.

Cultivating native bacteria from roots of plants grown in a given environment is essential for dissecting the functions of the root microbiota for plant growth and health with strain-specific resolution. In this study, we established a straightforward protocol for high-throughput bacterial isolation from fresh root samples using limiting dilution to ensure that most cultured bacteria originated from only one microorganism. This is followed by strain characterization using a two-sided barcode polymerase chain reaction system to identify pure and heterogeneous bacterial cultures. Our approach overcomes multiple difficulties of traditional bacterial isolation and identification methods, such as obtaining bacteria with diverse growth rates while greatly increasing throughput. To facilitate data processing, we developed an easy-to-use bioinformatic pipeline called 'Culturome' ( https://github.com/YongxinLiu/Culturome ) and a graphical user interface web server ( http://bailab.genetics.ac.cn/culturome/ ). This protocol allows any research group (two or three lab members without expertise in bioinformatics) to systematically cultivate root-associated bacteria within 8-9 weeks.