Table 2:
Overview of multi-modality single-cell epigenomics methods.
| Method | Single cell principle | Description |
|---|---|---|
| DNA methylation + RNA | ||
| scM&T-seq38 | plate-based, single cell dispension | Individual cells are sorted. After lysis, polyadenylated mRNA is captured using biotinylated oligo dT primers and separated from the DNA using strepatavidin-coupled magnetic beads. Single-cell full length transcriptome libraries and single cell bisulfite libraries are generated. |
| scMT-seq106 | tube-based, single cell dispension | Individual cells are picked and incubated in a drop of cell lysis buffer in a tube. After cell lysis, nuclei are transferred with a micro pipette to another tube. Single-cell full length transcriptome libraries for cytosolic RNA and scRRBS libraries are generated. |
| scTrio-seq107 and scTrio-seq108 | tube-based, single cell dispension | Individual cells are mouth pipetted into tubes and lysed. After cell lysis, nuclei are pelleted, and the supernatant is transferred to another tube. Single-cell transcriptome libraries for cytosolic RNA and scRRBS libraries are generated. Copy number variation (CNV) detection as third modality. |
| Chromatin accessibility + RNA | ||
| sciCAR109 | split-pool (2 rounds) | RNA is reverse transcribed with an indexed oligo(dT) primer followed by tagmentation with indexed Tn5. After one round of split-pooling and second strand synthesis, nuclei are lysed and separated for RNA and ATAC library preparation. The second index is introduced by PCR during library preparation. |
| SNARE-seq110 | droplet, custom | Nuclei are tagmented using Tn5 without index. Nuclei are encapsulated in droplets and a splint oligonucleotide is added to link tagmented DNA to oligo(dT) barcoded beads. After reverse transcription, separate libraries for cDNA and tagmented DNA are generated. |
| SNARE-seq2115 | split-pool (4 rounds) | Adaptation of SNARE-seq to combinatorial indexing. |
| Paired-Seq111 | split-pool (5 rounds) | Tagmentation precedes reverse transcription with sample specific indexes for both DNA and RNA. Barcodes are introduced through 3 rounds of ligation. After preamplification, product is split for DNA and RNA library preparation. DNA and RNA products are distinguished by molecule specific restriction enzyme sites. |
| SHARE-seq112 | split-pool (3 rounds) | Fixed nuclei are first tagmented and then reverse transcribed with a biotinylated indexed primer. Barcodes are introduced through 3 rounds of hybridization. After reverse crosslinking, cDNA is bound to streptavidin beads and separated from the supernatant containing tagmented DNA for library preparation. |
| ASTAR-seq113 | microfluidics chamber (Fluidigm) | Nuclei are first tagmented in the integrated fluidic circuit (IFC) and after quenching of the transposition RNA is reverse transcribed and cDNA amplified using biotinylated primers. Products are removed from the IFC and cDNA and tagmented DNA separated using streptavidin beads. |
| scCAT-seq114 | plate-based, single cell dispension | Cells are lysed, and the nucleus is separated from the cytoplasm by centrifugation. Separate libraries for chromatin accessibility and full-length transcriptomes are generated. |
| Chromium Single Cell Multiome ATAC + Gene Expression | droplet, 10x Genomics | Nuclei are tagmented using Tn5 without index. Nuclei are encapsulated into droplets with beads containing indexed adapters for both tagmented DNA and RNA. After breaking of the emulsion, tagmented DNA and cDNA are amplified by PCR and separated for library construction. |
| SNuBar-ARC61 | indexing + droplet (10x Genomics) | Tagmentation with Tn5. The index at the nuclei level is introduced using an oligonucleotide adapter during tagmentation that is complementary to the universal part of the transposon and contains a PCR handle, sample barcode and a polyA tail. |
| Histone modifications + RNA | ||
| Paired-Tag117 | split-pool (4 rounds) | This assay is a combination of CUT&Tag83 and Paired-Seq111. Histone modifications are targeted by antibody and indexed pA-Tn5 followed by indexed reverse transcription. After reverse transcription, nuclei are pooled and redistributed into new plates. After two more rounds of indexing by ligation, nuclei are pooled, preamplified and then split for RNA and DNA library preparation. DNA and RNA products are distinguished by molecule specific restriction enzyme sites. Profiled modifications: H3K4me1, H3K4me3, H3K27ac, H3K9me3, H3K27me3 |
| CoTECH116 | split-pool (2 rounds) | A modification of CoBATCH56 to enable simultaneous measurement of RNA. Histone modifications are targeted by antibody and indexed Protein-Tn5 followed by indexed reverse transcription. After reverse transcription, nuclei are pooled, redistributed into new plates and preamplified. After preamplification, products are split into two plates and a second set of indexes is introduced using indexed primers for DNA and RNA component, respectively. Profiled modifications: H3K4me3, H3K27ac, H3K27me3 |
| scSET-seq118 | plate-based, single cell dispension | Single cells are transferred to wells of a 96-well plate. Cells are lysed, and nuclei bound by Concanavalin A coated magnetic beads. The supernatant with RNA is transferred to another plate. Histone modifications are targeted by antibody and indexed pA-Tn5. Cytoplasmic RNA is reverse transcribed and mRNA/cDNA hybrids are tagmented with indexed Tn5. Profiled modifications: H3K4me3, H3K27me3 |
| Chromatin accessibility + protein | ||
| ASAP-seq120 | droplet, 10x Genomics | Cells are incubated with antibody-oligonucleotide conjugates against cell surface proteins. After fixation and permeabilization without lysis, cells are used as input for 10x scATAC with the addition of a bridge oligo that enables binding of the antibody-oligonucleotide conjugates to the index coated beads. |
| Pi-ATAC122 | plate-based, single cell dispension | Cells or tissues are fixed and permeabilized. Cells are incubated with an antibody and transposition is performed in bulk. Tagmentation is quenched and single cells are sorted into wells using index sorting to record antibody signal. Libraries are generated with indexed PCR primers after reverse crosslinking. |
| PHAGE-ATAC123 | droplet, 10x Genomics | Cells are incubated with nanobody-displaying phages to recognize and bind surface antigens. Cells are fixed, lysed and used as input for 10x scATAC. Phages express a PHAGE-ATAC tag (PAC-tag) that contains an Illumina Read1 sequence and a hypervariable genetic barcode that binds to the oligo coated beads in the droplets. Workflow is compatibe with ASAP-seq |
| Chromatin accessibility + RNA + protein | ||
| DOGMA-seq120 | droplet, 10x Genomics | Based on ASAP-seq but combined with 10x scMultiome instead of 10x scATAC. No need for bridge oligo. |
| TEA-seq121 | droplet, 10x Genomics | Cells are incubated with antibody-oligonucleotide conjugates against cell surface proteins. After permeabilization without lysis, cells are used as input for 10x scMultiome. |
| NEAT-seq125 | droplet, 10x Genomics | Cells are fixed, lysed and nuclei permeabilized. Nuclei are preincubated with ssDNA oligos and antibody oligo conjugates are preincubated with single stranded DNA from E coli (EcoSSB). Nuclei are incubated with antibodies against nuclear pore complex with hash tag oligos (HTO) and against transcription factors with antibody-derived tags (ADT). HTO and ADT contain a poly-A sequence. After incubation, nuclei are used as input for 10x scMultiome. |
| Histone modifications + Protein | ||
| scCUT&Tag-pro124 | droplet (10x Genomics) | Based on ASAP-seq120. Cells are incubated with oligonucleotide-conjugated antibodies against cell surface proteins. Cells are fixed and permeabilized without lysis. Cells are incubated with primary antibodies against a histone modification, followed by secondary antibodies and pA-Tn5. After tagmentation, cells are loaded onto the Chromium controller and libraries generated following the 10x scATAC-seq protocol. Profiled modifications: H3K4me1, H3K4me2, H3K4me3, H3K9me3, H3K27ac, H3K27me3 |
| DNA methylation + Chromatin accessibility | ||
| scNOMe-seq127 | plate-based, single cell dispension | Nuclei are isolated and treated in bulk with M.CviPI which methylates GpC dinucleotides at nucleosome free chromatin. Individual nuclei are sorted, lysed, bisulfite converted, and sequencing libraries are prepared. |
| scCOOL-seq126 and iscCOOL-seq128 | plate-based, single cell dispension | Nuclei are isolated and treated in bulk with M.CviPI which methylates GpC dinucleotides at nucleosome free chromatin. Individual nuclei are sorted, lysed, bisulfite converted, and sequencing libraries are prepared. In iscCOOL-seq a tailing- and ligation-free method for library prep is used to increase the mapping rates |
| DNA methylation + Chromatin accessibility + RNA | ||
| scNMT-seq129 | plate-based, single cell dispension | Nuclei are treated with M.CviPI which methylates GpC dinucleotides at nucleosome free chromatin. Libraries are generated following the scM&T-seq strateqy. |
| snmCAT-seq48 | plate-based, single cell dispension | Nuclei or cells are treated with M.CviPI to methylate GpC dinucleotides at nucleosome free chromatin. No physical separation of RNA and DNA. 5’-methyl-CTP is added during reverse transcription for full length transcriptomes. Bisulfite conversion and library preparation as in snmC-seq2. 5’-methyl-CTP in cDNA are not converted to uracil during bisulfite conversion, whereas a fraction of DNA cytosines is unmethylated and converted to uracil. Sequencing reads are assigned to RNA and DNA libraries based on the original mC density. |
| scNOMeRe-seq130 | tube-based, single cell dispension | Single cells are transferred to tubes by mouth pipetting. Cells are lysed and nuclei are bound to magnetic beads. Supernatant with RNA is transferred to another tube for library preparation using multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq) strategy. Nuclei are treated with M.CviPI to methylate GpC dinucleotides and lysed followed by bisulfite conversion and library preparation. |
| Heterochromatin + Chromatin accessibility | ||
| scGET-seq131 | droplet, 10x Genomics | Tn5 fused to chromodomain of HP-1α to target H3K9me3 regions (TnH). Nuclei are tagmented using Tn5 without index to target open chromatin followed by incubation with TnH to target H3K9me3 regions. Libraries are generated with 10x ATAC. Profiled modifications: H3K9me3 |
| Multiple histone modifications from the same cell | ||
| scMulti-CUT&Tag132 | Droplet, 10x Genomics | Adaptation of CUT&Tag83. Antibodies against distinct histone modifications are incubated with indexed pA-Tn5. Antibody-pA-Tn5 conjugates are purified and antibodies against two histone modifications are mixed. Samples are incubated with antibody-pA-Tn5 mixtures. After tagmentation, nuclei are encapsulated into gel emulsions on a Chromium controller and libraries are prepared following the 10x scATAC-seq protocol with slight modification. Profiled modifications: H3K27ac, H3K27me3 |
| DNA methylation + chromatin architecture | ||
| scmethylHiC133 | plate-based, single cell dispension | Crosslinking, restriction enyzme digestion and ligation are performed in bulk. Individual nuclei are sorted into wells (96--well plate). After bisulfite conversion libraries are generated. |
| snm3C-seq134 | plate-based, single cell dispension | Crosslinking, restriction enyzme digestion and ligation are performed in bulk. Single nuclei are sorted into wells (384-well plate). DNA is reverse crosslinked, and bisulfite converted. Libraries are generated using snmC-seq224. |
scM&T-seq, single-cell genome-wide methylome and transcriptome sequencing; scMT-seq, single-cell methylome and transcriptome sequencing; scTrio-seq, single-cell triple omics sequencing; sciCAR, single-cell combinatorial indexing chromatin accessibility and mRNA; SNARE-seq, single-nucleus chromatin accessibility and mRNA expression sequencing; Paired-Seq, parallel analysis of individual cells for RNA expression and DNA accessibility by sequencing; SHARE-seq, simultaneous high-throughput ATAC and RNA expression with sequencing; ASTAR-seq, assay for single-cell transcriptome and accessibility regions; scCAT-seq, single-cell chromatin accessibility and transcriptome sequencing; SNuBar-ARC, single nucleus barcoding approach for chromatin accessibility and RNA expression co-profiling; Paired-Tag, parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing; CoTECH, combined assay of transcriptome and enriched chromatin binding; scSET-seq, same cell epigenome and transcriptome sequencing in single cells; ASAP-seq, ATAC with select antigen profiling by sequencing, Pi-ATAC, protein-indexed assay of transposase accessible chromatin with sequencing; TEA-seq, transcription, epitopes, and accessibility with sequencing; NEAT-seq, sequencing of nuclear protein epitope abundance, chromatin accessibility and the transcriptome in single cells; scCUT&Tag-pro, single-cell cleavage under targets and tagmentation with cell surface proteins; scNOMe-seq, single- cell nucleosome occupancy and methylome-sequencing; scCOOL-seq, single-cell chromatin overall omic-scale landscape sequencing; iscCOOL-seq, improved single-cell chromatin overall omic-scale landscape sequencing; scNMT-seq, single-cell nucleosome, methylation and transcription sequencing; snmCAT-seq, single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing; scNOMeRe-seq, single-cell nucleosome occupancy, methylome and RNA expression sequencing; scGET-seq, single-cell genome and epigenome by transposases sequencing; scMethyl-HiC, single-cell DNA methylation with HiC, snm3C-seq, single-nucleus methyl-3C sequencing