Swanson, Elliott et al. “Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq.” eLife vol. 10 e63632. 9 Apr. 2021, doi:10.7554/eLife.63632

Tn5 transposase is currently a broadly applied biological tool in the field of genetic modification/testing. This popular product carried by Beta LifeScience has been applied to lots of academic research and has been mentioned over time as one of the accessible, fundamental tools among the field. The paper being referred to presents the development of two novel single-cell sequencing techniques, ICICLE-seq and TEA-seq, which allow for the simultaneous measurement of multiple cellular characteristics. These techniques were developed using human peripheral blood cells as a test case.

ICICLE-seq (Integrated Cellular Indexing of Chromatin Landscape and Epitopes) is a new scATAC-seq workflow that improves signal-to-noise and enables paired measurements of cell surface markers and chromatin accessibility. By integrating these measurements, researchers can better understand how genes are regulated in different cell types based on their surface markers.

TEA-seq (Transcriptomics, Epitopes, and Chromatin Accessibility sequencing) is a droplet-based multi omics platform that combines scRNA-seq, epitope measurements, and scATAC-seq to simultaneously analyze transcriptomics, cell surface markers, and chromatin accessibility in thousands of single cells. This trimodal assay provides a more comprehensive view of cellular characteristics, which can help identify type-specific gene regulation and expression based on phenotypically defined cell types.

Both ICICLE-seq and TEA-seq are innovative tools that can be used to study cell heterogeneity, differentiation, and cellular responses to various signals, ultimately contributing to a better understanding of human diseases.

ICICLE-seq requires the process of customizing oligonucleotide source. This process includes  Tn5 complexing, indexing, amplification, and ICICLE-seq library preparation.

In the Tn5 complexing protocol mentioned in the article, Tn5 transposase is used to form transposomes, which are complexes of the enzyme bound to the transposon DNA containing the sequencing adapters. Here's a step-by-step breakdown of the process:

• DNA complexes containing mosaic-end sequences with either a poly-T or Nextera R2N 5' overhang are created by annealing equimolar amounts of top and bottom oligos. This is done using a thermal cycler, which heats the sample to 95°C and then gradually cools it down to 20°C.
• Annealed DNA complexes are mixed 1:1 for a final concentration of 8 nM.
• Tn5 transposase is supplemented with 5 M NaCl at a final volume ratio of 1:8 NaCl to Tn5. This step is performed to stabilize the Tn5 enzyme and ensure optimal activity.
• The NaCl/Tn5 mixture is combined with the annealed DNA complexes at a volume ratio of 1.2:1 (DNA complexes to Tn5). This allows the Tn5 transposase to bind the DNA complexes and form transposomes.
• The mixture is incubated at 25°C for 60 minutes to ensure the formation of the final, reaction-ready Tn5 transposomes.
• The Tn5 transposomes are stored at -20°C until use.

In the context of scATAC-seq, these Tn5 transposomes will be used to fragment accessible chromatin regions in single cells and simultaneously insert sequencing adapters. This enables researchers to generate a genome-wide map of accessible chromatin regions for each individual cell in the sample.

The assembly of Tn5 transposomes, which are complexes of the Tn5 transposase enzyme and DNA fragments that have been modified with adapter sequences for DNA sequencing library preparation. The transposomes were created by annealing oligos with mosaic-end sequences with either a poly-T or Nextera R2N 5' overhang, then mixed with Tn5 transposase supplemented with NaCl and incubated at 25°C for 60 min. The resulting Tn5 complexes were stored at -20°C until use.

The transposition was performed on 20,000 permeabilized cells using Poly-T overhang Tn5 complexes, followed by the addition of cell barcodes and ADTs via GEM generation using 10x Genomics 3′ RNA beads and subsequent amplification. Libraries were then size-selected and split into two reactions, with ATAC and ADT fragments amplified separately using different indexed P7 primers. Finally, SPRIselect reagent cleanups were performed to remove large DNA fragments and unused primers.

With the pervasive use of Tn5 transposase in experiments which require insertion of DNA sequences into other DNA molecules, it’s now widely participating in biotechnology, genome engineering, library construction, and sequencing, allowing researchers to study the function of specific genes or regulatory regions. Additionally, Tn5 transposase can be used in library construction for high-throughput screening of genetic variants. It has also been used in sequencing technologies, such as Nextera sequencing, to prepare libraries for sequencing with high efficiency and low input DNA requirements. Overall, Tn5 transposase has become a powerful tool in biotechnology for its ability to facilitate DNA manipulation and analysis.

Reference:

Swanson, Elliott et al. “Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq.” eLife vol. 10 e63632. 9 Apr. 2021, doi:10.7554/eLife.63632