CHANGE-seq

CHANGE-seq (Circularization for High-throughput Analysis of Nuclease Genome-wide Effects by sequencing) is a method to study the effects of CRISPR-Cas9 off-target activity, which is crucial in the development of novel therapeutic approaches based on genome editing. In this method, Tagmentase (Tn5 transposase) with a custom oligonucleotide containing a 19 bp mosaic end and 4 palindromic bp with uracil is used to circularize the genome. This is followed by treatment with a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda, using different guide RNAs. ​.

Bestas, B., et al. A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo. Nat Commun 14, 5474 (2023). https://doi.org/10.1038/s41467-023-41240-7.

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​Tn5 Tagmentation-mediated integration site enrichment

DNA isolated from the host cells is fragmented using Tn5 transposase loaded with a single oligonucleotide containing a mosaic end and an Illumina adapter sequence (D.L. Stern, 2021). The library is prepared by PCR using primers that anneal to either end of the sequence, and a sequencing primer complementary to the Illumina adapter. Hologic Diagenode Tagmentase (Tn5 transposase) was used in this approach to determine whether SARS-CoV-2 can integrate into host genomes.​

Zhang L., et al. Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues. Proc Natl Acad Sci U S A. 2021 May 25;118(21):e2105968118. doi: 10.1073/pnas.2105968118. PMID: 33958444; PMCID: PMC8166107.

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​TRACE

TRACE (Transposase-Assisted Capture of Transposable Elements) enables the determination of the number of vector integration sites. The method involves tagmentation of purified genomic DNA using Tagmentase Tn5 transposase loaded with custom oligonucleotide. These oligonucleotides facilitate two rounds of nested PCR to amplify fragments containing junctions between the Sleeping Beauty transposase inverted repeats (IR) and adjacent DNA at sites of payload insertion. After library preparation and sequencing, the subsequent analysis identifies the locations of the insertions within the genome.​

Wang H. et al. Auto-expansion of in vivo HDAd-transduced hematopoietic stem cells by constitutive expression of tHMGA2, Molecular Therapy - Methods & Clinical Development, Volume 32, Issue 3, 2024,101319.

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​SMM-SV-seq

SMM-SV-seq (Single-Molecule Mutation Sequencing for Structural Variants) is capable of detecting single-nucleotide variants, small insertions, and deletions, as well as structural variants (SVs) such as deletions, duplications, insertions, inversions, and translocations. These SVs are particularly challenging to detect due to NGS library preparation artifacts such as chimeras. In this method, genomic DNA was tagmented with Tagmentase Tn5 transposase loaded with a custom oligonucleotide containing deoxyuridines. This allows the creation of 3’-ends that complement the adapters’ overhangs, enabling stringent ligation conditions that prevent illegitimate ligation of unprocessed DNA fragments and minimize chimeric artifacts indistinguishable from true SVs.

Detection of genome structural variation in normal cells and tissues by single molecule sequencing, Johanna Heid, Zhenqiu Huang, Moonsook Lee, Sergey Makhortov, Elizabeth Pan, Cristina Montagna, Shixiang Sun, Jan Vijg, Alexander Y. Maslov, bioRxiv 2024.08.08.607188; doi: https://doi.org/10.1101/2024.08.08.607188.

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​Transposon-directed insertion-site sequencing (TraDIS)

The incorporation of transposon or mini-transposon containing a selection marker (such as antibiotic resistance gene) to identify bacterial genes essential for growth under defined conditions was the first and, for a long time, the only application of the Tn5 enzyme. In the TraDIS approach, a sequence containing a kanamycin-resistant gene flanked by Mosaic Ends is loaded onto the Transposase, and the complex is electroporated into bacteria grown under defined conditions. Subsequently, a DNA library is prepared from the bacteria by enriching the Transposon-bacterial DNA junctions.

Kyono, Y., et al. Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS). Sci Rep 14, 6756 (2024). https://doi.org/10.1038/s41598-024-57537-6.​

sci-RNA-seq (Single Cell Combinatorial Indexing) technique and its modifications are capable of profiling the transcriptomes of individual cells. This method utilizes split-pool barcoding of nucleic acids within a single cell. Individual nuclei are fixed and sorted into separate wells of plates. Three indexes are added to mRNA/cDNA through reverse transcription, ligation, and tagmentation, respectively. During the tagmentation step, Tagmentase Tn5 transposase is loaded with a single i5 adaptor. Each step is followed by pooling and redistributing the samples into new plates to ensure a combinatorial distribution of indexes. This method is powerful as it can distinguish the transcriptomic profiles of a large number of cells without the need to isolate individual cells.

Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq)

Martin, B.K., et al. Optimized single-nucleus transcriptional profiling by combinatorial indexing. Nat Protoc 18, 188–207 (2023). https://doi.org/10.1038/s41596-022-00752-0.

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​Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3)

Li H., et al. Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing. STAR Protoc. 2022 Dec 16;3(4):101904. doi: 10.1016/j.xpro.2022.101904. Epub 2022 Dec 5. PMID: 36595916; PMCID: PMC9732400.

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T-RHEX-RNAseq

T-RHEX-RNAseq is a method that generates stranded RNA-seq libraries directly from very low numbers of lysed cells. This is achieved by creating an RNA library directly within the cell through simultaneous rRNA depletion and priming with random hexamers. Stranded information is obtained by incorporating uracils during the second strand cDNA synthesis. Subsequently, i5 adaptors are inserted using Tagmentase Tn5 transposase loaded with the respective adaptor, while i7 adaptors are added by primer annealing. The library is then amplified using Phusion polymerase, which is incompatible with dUTP, thereby facilitating the generation of stranded libraries.

Gustafsson C., et al. T-RHEX-RNAseq - a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells. BMC Genomics. 2023 Apr 17;24(1):205. doi: 10.1186/s12864-023-09279-4. PMID: 37069502; PMCID: PMC10111750.​

ATAC-seq

ATAC-seq (Assay for Transposase-Accessible Chromatin, followed by next-generation sequencing) is one of the most well-cited and widely used techniques that utilize the ability of Tn5 to cut DNA in its chromatin form. This feature leverages the ability of the Tn5 transposase to understand the landscape of chromatin accessibility in the cell. In this technique, Tagmentase Tn5 transposase, loaded with standard Illumina-compatible adaptors, is incubated with permeabilized cells or nuclei, where it cuts out fragments of open chromatin. This method is rapid and compatible with low input and various sample types.​

Different modifications of this approach utilize not only the overall method features but also the convenience of the modification of the oligos complexed with Tn5.​

Miyazaki S., et al. Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells. Commun Biol. 2023 Jul 24;6(1):771. doi: 10.1038/s42003-023-05148-8. PMID: 37488353; PMCID: PMC10366109.​

Yamazaki M., et al. YAP/BRD4-controlled ROR1 promotes tumor-initiating cells and hyperproliferation in pancreatic cancer. EMBO J. 2023 Jul 17;42(14):e112614. doi: 10.15252/embj.2022112614. Epub 2023 Apr 25. PMID: 37096681; PMCID: PMC10350825.​​

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ATAC-see

ATAC-see (Assay of Transposase-Accessible Chromatin with visualization) is a novel technique for visualizing accessible chromatin regions and accessing their sequence. Tagmentase Tn5 transposase is loaded with fluorophore-conjugated oligonucleotides. Fixed cells are tagmented before further visualization. After visualization, it is possible to quench the staining and release the tagged DNA fragments for sequencing. ​

Miyanari Y. Imaging Chromatin Accessibility by Assay of Transposase-Accessible Chromatin with Visualization. Methods Mol Biol. 2023;2577:93-101. doi: 10.1007/978-1-0716-2724-2_7. PMID: 36173568.

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​Spatial-ATAC-seq

While the ATAC-see method is more tailored for individual cells, spatial ATAC-seq is designed for in situ profiling of tissue samples. This allows for the observation of differences in chromatin accessibility across various cell types and reflects different physiological and developmental conditions. The spatial ATAC-seq setup utilizes Hologic Diagenode Tagmentase Tn5 transposase loaded with custom adaptors to tagment chromatin in an entire fixed tissue slice. Barcodes are then added using a microfluidics setup, with each barcode labeling a square in a 50x50 grid. The tagmented and barcoded DNA is subsequently used to read out chromatin accessibility in each of the 2,500 squares and the whole sample.​

Deng Y., et al. Spatial profiling of chromatin accessibility in mouse and human tissues. Nature. 2022 Sep;609(7926):375-383. doi: 10.1038/s41586-022-05094-1. Epub 2022 Aug 17. PMID: 35978191; PMCID: PMC9452302.

Spatial ATAC-RNA-seq

ATAC-seq profiling is a method that is done on native, permeabilized cells, allowing the execution of other techniques in parallel while understanding chromatin accessibility. Spatial ATAC-RNA-seq is a logical continuation of the Spatial ATAC-seq technology. Using a similar tagmentation approach, supplemented with the addition of a poly-T-biotinylated adaptor oligo to capture mRNA followed by barcoding, it became possible to simultaneously profile both epigenomic and transcriptomic features of mouse and human tissues.

Zhang, D., et al. Spatial epigenome–transcriptome co-profiling of mammalian tissues. Nature 616, 113–122 (2023). https://doi.org/10.1038/s41586-023-05795-1.

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SHARE-seq

SHARE-seq (Simultaneous High-Throughput ATAC and RNA Expression with sequencing) is a similar approach for simultaneous transcriptomic and epigenomic profiling that follows a similar order of events. First, fixed and permeabilized cells (or nuclei) are tagmented with Tagmentase Tn5 transposase loaded with Illumina-compatible adaptors to release open chromatin fragments. Then, mRNA is reverse-transcribed with poly-T primer with a biotin tag. Both DNA fragments and the resulting cDNA are barcoded, separated into respective pools with streptavidin beads, and used for library preparation.

Scholz, R., et al. Combined Analysis of mRNA Expression and Open Chromatin in Microglia. In: Mass, E. (eds) Tissue-Resident Macrophages. Methods in Molecular Biology, vol 2713. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3437-0_35.

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scGET-seq

scGET-seq (Single-Cell Genome and Epigenome by Transposases sequencing) simultaneously profiles euchromatin and heterochromatin. scGET-seq exploits the concurrent action of transposase Tn5 and its hybrid form TnH, which targets H3K9me3 domains. This means that it utilizes the ability to modify not only the oligonucleotides complexed with Tn5, but also the Tn5 enzyme itself (similarly to the pA-Tn5 enzyme – add a link to the product page here). scGET provides a unique perspective on chromatin changes in individual cells.​

Cittaro D., et al. Analyzing genomic and epigenetic profiles in single cells by hybrid transposase (scGET-seq). STAR Protoc. 2023 Mar 29;4(2):102176. doi: 10.1016/j.xpro.2023.102176. Epub ahead of print. PMID: 37000619; PMCID: PMC10090441.