The ICELL8 cx Single-Cell System allows you to maximize your biomarker discovery with combinatorial indexing

Advances in chromatin profiling technologies have helped uncover gene regulatory features and critical biomarkers from single-cell samples. However, such assays remain expensive and challenging due to the lack of precise and scalable methods. Recently, two groups have published exciting research breakthroughs showing how they leveraged the open and flexible ICELL8 cx Single-Cell System to close this gap.

Combinatorial indexing

Multimodal profiling of chromatin features

Scientists from Steven Henikoff’s lab at Fred Hutch Cancer Center developed an approach called Multiple Target Identification by Tagmentation (MulTI-Tag) for physical association of a chromatin protein-targeting antibody with an identifying adapter barcode (Meers et al. 2023). The adapter barcode is added via tagmentation, a process using hyperactive transposase to rapidly and simultaneously fragment DNA and tag the fragments using adapter sequences, which can demystify different histone modification landscapes at single-cell resolution. They further optimized this assay with combinatorial indexing, leading to an exponential increase in cell throughput.

In the MulTI-Tag assay, antibody-oligonucleotide conjugates are used to physically associate forward-adapter barcodes with genomic targets of interest. They are loaded directly onto pA-Tn5 transposomes for sequential binding and tagmentation (Figure 1, Step 1). Meanwhile, pA-Tn5 loaded only with reverse adapters are used for a secondary CUT&Tag step (Figure 1, Step 2) to efficiently introduce the reverse adapters into genomic loci bound by conjugates during Step 1.

To scale up the workflow, the Henikoff lab scientists used MulTI-Tag to profile H1 hESCs and K562 cells using antibodies against H3K27me3 and H3K36me3, separately or in a cell mixture. Afterwards, they dispensed the cells into the 350 nl blank chips with the ICELL8 system and attributed combinatorial barcodes via PCR amplification (Figure 2a). After Illumina sequencing, they generated MulTI-Tag libraries from a total of 21,548 cells with unique reads for H3K27me3 and H3K36me3. At least 80% of these unique fragments overlapped with the expected targets defined by ENCODE ChIP-seq (Figure 2b). Other QC metrics, such as uniformity of coverage, cell recovery from input, and library complexity, were all similar or even superior to other published methods. The results from this study demonstrate MulTI-Tag as a step forward in probing chromatin regulation at the single-cell level.

Atlas-scale measurement of chromatin accessibility at low cost

In another paper recently published in Genome Research (O'Connell et al. 2023), scientists from Andrew Adey’s lab at Oregon Health & Science University (OHSU) leveraged the ICELL8 cx Single-Cell System to achieve an over 50-fold improvement in scale while greatly reducing library prep cost per cell compared to canonical single-cell ATAC-seq (scATAC-seq) assays.

With scATAC-seq widely used to depict open chromatin regions, scientists have sought different technological improvements to increase cell throughput and lower reagent costs. Major strategies involve in situ tagmentation followed by cell indexing during PCR in droplets (e.g. 10x Chromium system) or combinatorial indexing to add multiple rounds of barcoding indexes in 96-well plates (sciATAC). sciATAC is preferred by many researchers as it does not require individual reactions for each cell, eliminating the need for handling a large number of 96-well plates.

In this study, Dr. Adey’s group went above and beyond current methods, overcoming the major limitation of tedious pipetting steps by using a multi-sample nano-dispenser, which can automatically fill up to 5,184 nanowells with a reagent master mix. In addition, they leveraged the 350 nl blank chip and the flexibility of the ICELL8 cx system to carry out the PCR round of barcoding at nanoliter scale as part of the new sciATACv3 workflow, which improves tagmentation efficiency by allowing shorter adapter sequences to be used. The design includes an index on only one of the adapter molecules that is adjacent to the transposon recognition sequence, as opposed to both the forward and reverse adapters. The lab innovatively combined the advantages of  combinatorial indexing and nanowell chips to obtain an over 50-fold improvement in cell throughput (5,184 nanowells vs 96-well plate) and realized a huge per-cell cost savings since the nanowell chip uses approximately the same PCR reagents amounts as a single 96-well plate PCR reaction (Figure 3a).

Moreover, in sciATACv3, they also made significant optimization to the sciATAC chemistry and achieved a substantial enhancement in data quality by minimizing crosstalk and doublet rates. When benchmarked with annotated DHS (DNase I hypersensitivity) peaks identified in the ENCODE ChIP-seq project, data generated from nanowell-based sciATACv3 workflow showed a higher median fraction of reads in peaks (FRiP) than scATAC-seq data acquired with droplet-based methods, suggesting more reliable and accurate deconvolution of open chromatin regions at single-cell resolution (Figure 3b). Taken together, the results of this study demonstrate that the new sciATACv3 workflow provides similar or superior per-cell information at scale while reducing costs compared to current canonical assays.

Summary

Combinatorial indexing, a powerful tool for atlas-scale single-cell studies, has been more and more widely used and combined with different chemistries to identify crucial transcriptomic, genomic, and epigenomic biomarkers at single-cell level. However, all combinatorial indexing methods commercially available nowadays rely on tedious pipetting in a large number of microwell plates, making them susceptible to human errors. The open and flexible ICELL8 cx system, along with its super accurate nanoliter-scale dispensing capability, perfectly addresses this problem and unleashes the potential to add 5,000+ unique barcoding index sequences per round. The recent successes of these customers from Fred Hutch and OHSU not only demonstrated how ICELL8 cx can be utilized in combinatorial indexing assays for exponential increase of cell throughput, but also how cost per cell can be substantially lowered.

Combinatorial indexing without the need to pipette multiple microwell plates used to be in the ideal Utopia. Now it has become a reality with the ICELL8 cx Single-Cell System.

References

Meers, M.P., Llagas, G., Janssens, D.H. et al. Multifactorial profiling of epigenetic landscapes at single-cell resolution using MulTI-Tag. Nat. Biotechnol. 41, 708–716 (2023). https://doi.org/10.1038/s41587-022-01522-9

O'Connell, B.L., Nichols, R.V., Pokholok, D. et al. Atlas-scale single-cell chromatin accessibility using nanowell-based combinatorial indexing. Genome Res. 33, 208–217 (2023). https://doi.org/10.1101/gr.276655.122