Epigenomic Profiling of B Cell Subsets by CUT&Tag.

Epigenetic programs play a key role in regulating the development and function of immune cells. However, conventional methods for profiling epigenetic mechanisms, such as the post-translational modifications to histones, present several technical challenges that prevent a complete understanding of gene regulation. Here, we provide a detailed protocol of the Cleavage Under Targets and Tagmentation (CUT&Tag) chromatin profiling technique for identifying histone modifications in human and mouse lymphocytes.

Measuring B Cell Chromatin Accessibility by One-Step ATAC-seq.

The Assay for Transposase Accessible Chromatin (ATAC)-seq protocol is optimized to generate global maps of accessible chromatin using limited cell inputs. The Tn5 transposase tagmentation reaction simultaneously fragments and tags the accessible DNA with Illumina Nextera sequencing adapters. Fragmented and adapter tagged DNA is then purified and PCR amplified with dual indexing primers to generate a size-specific sequencing library. The One-Step workflow below outlines the Tn5 nuclei transposition from a range of cell inputs followed by PCR amplification to generate a sequencing library.

Transposase-Assisted RNA/DNA Hybrid Co-Tagmentation for Target Meta-Virome of Foodborne Viruses.

Foodborne diseases are major public health problems globally. Metagenomics has emerged as a widely used tool for pathogen screening. In this study, we conducted an updated Tn5 transposase-assisted RNA/DNA hybrid co-tagmentation (TRACE) library construction approach. To address the detection of prevalent known foodborne viruses and the discovery of unknown pathogens, we employed both specific primers and oligo-T primers during reverse transcription. The method was validated using clinical samples confirmed by RT-qPCR and compared with standard RNA-seq library construction methods. The mapping-based approach enabled the retrieval of nearly complete genomes (>95%) for the majority of virus genome segments (86 out of 88, 97.73%), with a mean coverage depth of 21,494.53× (ranging from 77.94× to 55,688.58×). Co-infection phenomena involving prevalent genotypes of Norovirus with Astrovirus and Human betaherpesvirus 6B were observed in two samples. The updated TRACE-seq exhibited superior performance in viral reads percentages compared to standard RNA-seq library preparation methods. This updated method has expanded its target pathogens beyond solely Norovirus to include other prevalent foodborne viruses. The feasibility and potential effectiveness of this approach were then evaluated as an alternative method for surveilling foodborne viruses, thus paving the way for further exploration into whole-genome sequencing of viruses.

Genetic Loci of Plant Pathogenic Dickeya solani IPO 2222 Expressed in Contact with Weed-Host Bittersweet Nightshade (Solanum dulcamara L.) Plants.

Dickeya solani, belonging to the Soft Rot Pectobacteriaceae, are aggressive necrotrophs, exhibiting both a wide geographic distribution and a wide host range that includes many angiosperm orders, both dicot and monocot plants, cultivated under all climatic conditions. Little is known about the infection strategies D. solani employs to infect hosts other than potato (Solanum tuberosum L.). Our earlier study identified D. solani Tn5 mutants induced exclusively by the presence of the weed host S. dulcamara. The current study assessed the identity and virulence contribution of the selected genes mutated by the Tn5 insertions and induced by the presence of S. dulcamara. These genes encode proteins with functions linked to polyketide antibiotics and polysaccharide synthesis, membrane transport, stress response, and sugar and amino acid metabolism. Eight of these genes, encoding UvrY (GacA), tRNA guanosine transglycosylase Tgt, LPS-related WbeA, capsular biosynthesis protein VpsM, DltB alanine export protein, glycosyltransferase, putative transcription regulator YheO/PAS domain-containing protein, and a hypothetical protein, were required for virulence on S. dulcamara plants. The implications of D. solani interaction with a weed host, S. dulcamara, are discussed.

Cut&tag: a powerful epigenetic tool for chromatin profiling.

Analysis of transcription factors and chromatin modifications at the genome-wide level provides insights into gene regulatory processes, such as transcription, cell differentiation and cellular response. Chromatin immunoprecipitation is the most popular and powerful approach for mapping chromatin, and other enzyme-tethering techniques have recently become available for living cells. Among these, Cleavage Under Targets and Tagmentation (CUT&Tag) is a relatively novel chromatin profiling method that has rapidly gained popularity in the field of epigenetics since 2019. It has also been widely adapted to map chromatin modifications and TFs in different species, illustrating the association of these chromatin epitopes with various physiological and pathological processes. Scalable single-cell CUT&Tag can be combined with distinct platforms to distinguish cellular identity, epigenetic features and even spatial chromatin profiling. In addition, CUT&Tag has been developed as a strategy for joint profiling of the epigenome, transcriptome or proteome on the same sample. In this review, we will mainly consolidate the applications of CUT&Tag and its derivatives on different platforms, give a detailed explanation of the pros and cons of this technique as well as the potential development trends and applications in the future.

Selection of Flax Genotypes for Pan-Genomic Studies by Sequencing Tagmentation-Based Transcriptome Libraries.

Flax (Linum usitatissimum L.) products are used in the food, pharmaceutical, textile, polymer, medical, and other industries. The creation of a pan-genome will be an important advance in flax research and breeding. The selection of flax genotypes that sufficiently cover the species diversity is a crucial step for the pan-genomic study. For this purpose, we have adapted a method based on Illumina sequencing of transcriptome libraries prepared using the Tn5 transposase (tagmentase). This approach reduces the cost of sample preparation compared to commercial kits and allows the generation of a large number of cDNA libraries in a short time. RNA-seq data were obtained for 192 flax plants (3-6 individual plants from 44 flax accessions of different morphology and geographical origin). Evaluation of the genetic relationship between flax plants based on the sequencing data revealed incorrect species identification for five accessions. Therefore, these accessions were excluded from the sample set for the pan-genomic study. For the remaining samples, typical genotypes were selected to provide the most comprehensive genetic diversity of flax for pan-genome construction. Thus, high-throughput sequencing of tagmentation-based transcriptome libraries showed high efficiency in assessing the genetic relationship of flax samples and allowed us to select genotypes for the flax pan-genomic analysis.

Enhancing polyhydroxyalkanoate production in Cupriavidus sp. L7L through wcaJ gene deletion.

Cupriavidus sp. L7L synthesizes a high content of ductile polyhydroxyalkanoate. However, during fermentation, the medium's viscosity gradually increases, eventually reaching a level similar to 93 % glycerol, leading to fermentation termination and difficulties in cell harvest. A non-mucoid variant was isolated from a mini-Tn5 mutant library with the transposon inserted at the promoter sequence upstream of the wcaJ gene. Deletion of wcaJ eliminated the mucoid-colony appearance. The complementation experiment confirmed the association between wcaJ gene expression and mucoid-colony formation. Additionally, the wild-type strain exhibited a faster specific growth rate than the deletion strain using levulinate (Lev) as a carbon source. In fed-batch fermentation, Cupriavidus sp. L7L∆wcaJ showed similar PHA content and monomer composition to the wild-type strain. However, the extended fermentation time resulted in a 42 % increase in PHA concentration. After fed-batch fermentation, the deletion strain's medium had only 8.75 % of the wild-type strain's extracellular polymeric substance content. Moreover, the deletion strain's medium had a much lower viscosity (1.04 mPa·s) than the wild-type strain (194.7 mPa·s), making bacterial cell collection easier through centrifugation. In summary, Cupriavidus sp. L7L∆wcaJ effectively addressed difficulties in cell harvest, increased PHA production, and Lev-to-PHA conversion efficiency, making these characteristics advantageous for industrial-scale PHA production.

HiCAR: Analysis of Open Chromatin Associated Long-range Chromatin Interaction Using Low-Input Materials.

Cis-regulatory elements (cREs) and their long-range interactions are crucial for spatial-temporal gene regulation. While cREs can be characterized as accessible chromatin sequences, comprehensively identifying their spatial interactions remains a challenge. We recently developed a method, HiCAR (Hi-C on Accessible Regulatory DNA), which combines Tn5 transposase and chromatin proximity ligation to analyze open chromatin-anchored interactions in low-input cells. Application of HiCAR in human embryonic stem cells and lymphoblastoid cells reveals high-resolution chromatin contacts with efficiency comparable to in situ Hi-C across various distance ranges. Moreover, HiCAR was successfully applied to 30,000 primary human muscle stem cells, showcasing its potential for analyzing chromatin accessibility and looping in low-input primary cells and clinical samples. Here, we provide a detailed step-by-step protocol to perform the updated HiCAR experiments. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Tn5 Transposase Assembly Basic Protocol 2: HiCAR Library Preparation.

Identification and Characterization of Some Genes, Enzymes, and Metabolic Intermediates Belonging to the Bile Acid Aerobic Catabolic Pathway from Pseudomonas.

The study of the catabolic potential of microbial species isolated from different habitats has allowed the identification and characterization of bacteria able to assimilate bile acids and/or other steroids (e.g., testosterone and 4-androsten-3,17-dione) under aerobic conditions through the 9,10-seco pathway. From soil samples, we have isolated several strains belonging to genus Pseudomonas that grow efficiently in chemically defined media containing some cyclopentane-perhydrophenanthrene derivatives as carbon sources. Genetic and biochemical studies performed with one of these bacteria (P. putida DOC21) allowed the identification of the genes and enzymes belonging to the route involved in bile acids and androgens, the 9,10-seco pathway in this bacterium. In this manuscript, we describe the most relevant methods used in our lab for the identification of the chromosomal location and nucleotide sequence of the catabolic genes (or gene clusters) encoding the enzymes of this pathway, and the tools useful to establish the role of some of the enzymes that participate in this route.

(Tn5-)FISH-based imaging in the era of 3D/spatial genomics.

3D genomics mainly focuses on the 3D position of single genes at the cell level, while spatial genomics focuses more on the tissue level. In this exciting new era of 3D/spatial genomics, half-century old FISH and its derivative methods, including Tn5-FISH, play important roles. In this review, we introduce the Tn5-FISH we developed recently, and present six different applications published by our collaborators and us, based on (Tn5-)FISH, which can be either general BAC clone-based FISH or Tn5-FISH. In these interesting cases, (Tn5-)FISH demonstrated its vigorous ability of targeting sub-chromosomal structures across different diseases and cell lines (leukemia, mESCs (mouse embryonic stem cells), and differentiation cell lines). Serving as an effective tool to image genomic structures at the kilobase level, Tn5-FISH holds great potential to detect chromosomal structures in a high-throughput manner, thus bringing the dawn for new discoveries in the great era of 3D/spatial genomics.

Tn5 Transposon-based Mutagenesis for Engineering Phage-resistant Strains of Escherichia coli BL21 (DE3).

Escherichia coli is a preferred strain for recombinant protein production, however, it is often plagued by phage infection during experimental studies and industrial fermentation. While the existing methods of obtaining phage-resistant strains by natural mutation are not efficient enough and time-consuming. Herein, a high-throughput method by combining Tn5 transposon mutation and phage screening was used to produce Escherichia coli BL21 (DE3) phage-resistant strains. Mutant strains PR281-7, PR338-8, PR339-3, PR340-8, and PR347-9 were obtained, and they could effectively resist phage infection. Meanwhile, they had good growth ability, did not contain pseudolysogenic strains, and were controllable. The resultant phage-resistant strains maintained the capabilities of producing recombinant proteins since no difference in mCherry red fluorescent protein expression was found in phage-resistant strains. Comparative genomics showed that PR281-7, PR338-8, PR339-3, and PR340-8 mutated in ecpE, nohD, nrdR, and livM genes, respectively. In this work, a strategy was successfully developed to obtain phage-resistant strains with excellent protein expression characteristics by Tn5 transposon mutation. This study provides a new reference to solve the phage contamination problem.

TRACE-seq: Rapid, Low-Input, One-Tube RNA-seq Library Construction Based on Tagmentation of RNA/DNA Hybrids.

Tn5 transposase has been widely used to simultaneously fragment and tag double-stranded DNA (dsDNA) with sequencing adaptors in library construction for next-generation sequencing. Recently, we demonstrated that Tn5 transposase also possesses tagmentation activity toward RNA/DNA hybrids, in addition to its canonical dsDNA substrates. Based on this new activity, we are able to skip multiple laborious and time-consuming steps in traditional RNA-seq methods, and rapid, low-input, cost-effective, one-tube RNA-seq library construction is thus enabled. The libraries constructed by Transposase-assisted RNA/DNA hybrids Co-tagmEntation (termed "TRACE-seq") demonstrate excellent performance in terms of gene expression measurement and differential gene expression analysis. Here, we present detailed protocols for TRACE-seq that will be broadly useful for the study of RNA biology and biomedical research. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Total RNA preparation Basic Protocol 2: TRACE-seq library construction Support Protocol: Tn5 transposome assembly.

Phage resistance of Salmonella enterica obtained by transposon Tn5-mediated SefR gene silent mutation.

Salmonella enterica contamination is a primary cause of global food poisoning. Using phages as bactericidal alternatives to antibiotics could confront the issue of drug resistance. However, the problem of phage resistance, especially mutant strains with multiple phage resistance, is a critical barrier to the practical application of phages. In this study, a library of EZ-Tn5 transposable mutants of susceptible host S. enterica B3-6 was constructed. After the infestation pressure of a broad-spectrum phage TP1, a mutant strain with resistance to eight phages was obtained. Analysis of the genome resequencing results revealed that the SefR gene was disrupted in the mutant strain. The mutant strain displayed a reduced adsorption rate of 42% and a significant decrease in swimming and swarming motility, as well as a significantly reduced expression of the flagellar-related FliL and FliO genes to 17% and 36%, respectively. An uninterrupted form of the SefR gene was cloned into vector pET-21a (+) and used for complementation of the mutant strain. The complemented mutant exhibited similar adsorption and motility as the wild-type control. These results suggest that the disrupted flagellar-mediated SefR gene causes an adsorption inhibition, which is responsible for the phage-resistant phenotype of the S. enterica transposition mutant.

Tn5 DNA Transposase in Multi-Omics Research.

Tn5 transposase use in biotechnology has substantially advanced the sequencing applications of genome-wide analysis of cells. This is mainly due to the ability of Tn5 transposase to efficiently transpose DNA essentially randomly into any target DNA without the aid of other factors. This concise review is focused on the advances in Tn5 applications in multi-omics technologies, genome-wide profiling, and Tn5 hybrid molecule creation. The possibilities of other transposase uses are also discussed.

Determination of the Chromatin Openness in Bacterial Genomes.

The hyperactive Tn5 transposase in the ATAC-seq method has been widely used to determine the open DNA regions and understand the overall epigenomic regulation in the chromatins of eukaryotic cells. Here, we describe POP-seq (Prokaryotic chromatin Openness Profiling sequencing), an adaptation of the ATAC-seq method, to interrogate changes in the openness of prokaryotic nucleoids.

Analysis of Chromatin Interaction and Accessibility by Trac-Looping.

Spatial organization of the genome modulates pivotal biological processes. The emerging new technologies have provided novel insights into genome structure and its role in regulating cell activities. To examine the genome-wide chromatin interactions at accessible chromatin regions, we developed a DNA transposase-mediated analysis of chromatin looping (Trac-looping) method for simultaneously detecting chromatin interactions and chromatin accessibility. Here, we describe a detailed protocol of generating Trac-looping libraries.

ATAC-See: A Tn5 Transposase-Mediated Assay for Detection of Chromatin Accessibility with Imaging.

Assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that enables direct imaging of the accessible genome in situ and deep sequencing to reveal the identity of the imaged elements. Here we image spatial organization of the accessible genome in HT1080 cells with this method.

Biotinylated Tn5 transposase-mediated CUT&Tag efficiently profiles transcription factor-DNA interactions in plants.

In contrast to CUT&Tag approaches for profiling bulk histone modifications, current CUT&Tag methods for analysing specific transcription factor (TF)-DNA interactions remain technically challenging due to TFs having relatively low abundance. Moreover, an efficient CUT&Tag strategy for plant TFs is not yet available. Here, we first applied biotinylated Tn5 transposase-mediated CUT&Tag (B-CUT&Tag) to produce high-quality libraries for interrogating TF-DNA interactions. B-CUT&Tag combines streptavidin-biotin-based DNA purification with routine CUT&Tag, optimizing the removal of large amounts of intact chromatin not targeted by specific TFs. The biotinylated chromatin fragments are then purified for construction of deep sequencing libraries or qPCR analysis. We applied B-CUT&Tag to probe genome-wide DNA targets of Squamosa promoter-binding-like protein 9 (SPL9), a well-established TF in Arabidopsis; the resulting profiles were efficient and consistent in demonstrating its well-established target genes in juvenile-adult transition/flowering, trichome development, flavonoid biosynthesis, wax synthesis and branching. Interestingly, our results indicate functions of AtSPL9 in modulating growth-defence trade-offs. In addition, we established a method for applying qPCR after CUT&Tag (B-CUT&Tag-qPCR) and successfully validated the binding of SPL9 in Arabidopsis and PHR2 in rice. Our study thus provides a convenient and highly efficient CUT&Tag strategy for profiling TF-chromatin interactions that is widely applicable to the annotation of cis-regulatory elements for crop improvement.

Uncovering the determinants of model Escherichia coli strain C600 susceptibility and resistance to lytic T4-like and T7-like phage.

As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance.

A novel strand-specific RNA-sequencing protocol using dU-adaptor-assembled Tn5.

Strand-specific RNA-seq is a powerful tool for the discovery of novel transcripts, annotation of genomes, and profiling of gene expression levels. Tn5 transposase has been successfully applied in massive-scale sequencing projects; in particular, Tn5 adaptor modification is used in epigenetics, genomic structure, and chromatin visualization. We developed a novel dU-adaptor-assembled Tn5-mediated strand-specific RNA-sequencing protocol and compared this method with the leading dUTP method in terms of experimental procedure and multiple quality metrics of the generated libraries. The results showed that the dU-Tn5 method is easy to operate and generates a strand-specific RNA-seq library of comparable quality considering library complexity, strand-specificity, evenness, and continuity of annotated transcript coverage. We also evaluated the performance of the dU-Tn5 method in identifying nitrogen-responsive protein-coding genes and long non-coding RNAs in soybean roots. The results indicated that ~62-70% of differentially expressed genes detected from conventional libraries were also detected in dU-Tn5 libraries, indicating good agreement of our method with the current standard; moreover, their fold-changes were highly correlated (R>0.9). Thus, our method provides a promising 'do-it-yourself' stranded RNA-seq procedure for gene expression profiling.