Table 1:
Overview of single modality single-cell epigenomics methods.
| Method | Single cell principle | Description |
|---|---|---|
| DNA methylation (5mC) | ||
| scBS-seq39 | tube-based, single cell dispension | Individual cells are distributed, lysed and treated with bisulfite. Fragments are captured by several rounds of random priming. Primed fragments are amplified by index PCR. |
| scWGBS40 | plate-based, single cell dispension | Individual cells are added to wells, lysed and treated with bisulfite. Single stranded bisulfite-converted DNA is transcribed using tagged random hexamers and subsequently 3’ ends are also tagged. Tagged fragments are amplified by PCR to introduce sequencing adapters. |
| snmCseq41 and snmC-seq242 | plate-based, single cell dispension | Single nuclei are sorted into plates. Bisulfite conversion is carried out prior to indexing by random priming and extension. Libraries are generated using an adapter, compatible with single-stranded, bisulfite-converted DNA. SnmC-seq2 increases data quality by using a different random primer and inactivation of nucleotide triphosphates after extension. Automation of snmC-seq2 allows processing of 3,072 (eight 384 well plates) per experiment. |
| scRRBS45 | tube-based, single cell dispension | Cells are picked and transferred to a tube. DNA is digested by methylation insensitive Mspl to enrich for CG rich regions (for example, CpG islands) of the genome. After end repair and adapter ligation, DNA is bisulfite converted, PCR amplified, and size selected for sequencing. |
| scXRBS46 | plate-based, single cell dispension | Cells are sorted into individual wells. DNA is digested with Mspl and ligated to an indexed biotinylated adapter. After ligation wells are pooled and bisulfite conversion is performed on the pool. The second adapter is introduced using random hexamer extension prior to PCR amplification. Random hexamers help to rescue degraded fragments and enable measuring of regions with an isolated Mspl site. |
| sci-MET47 | split-pool (2 rounds) | Nuclei from crosslinked cells are distributed into wells and tagmentation is performed with indexed Tn5 without cytosines. After tagmentation nuclei are pooled, redistributed and bisulfite converted. After random priming and linear amplification, the second barcode is introduced by index PCR. |
| Chromatin accessibility | ||
| scDNAse-seq50 | plate-based, single cell dispension | Individual cells are sorted, and chromatin is digested with DNase I. Circular carrier DNA is added before end repair and adaptor ligation to minimize loss of digested fragments. Cells are indexed by PCR. |
| iscDNase-seq51 | split-pool (3 rounds) | DNase I digestion in bulk cells. Indexed P7 adaptor ligated in 96-well plates in bulk. After pooling and redistributing another index is introduced using PCR and finally the whole sample is marked by a third index introduced by PCR. |
| scATAC-seq52,53 | plate-based, single cell dispension | Nuclei are isolated and tagmented in bulk. Nuclei are sorted, lysed, and released DNA fragments are PCR amplified using indexed primers. |
| scATAC-seq54,55 | Strip-tubes/plates, single cell dispension | Individual nuclei are sorted into wells prior to tagmentation. Tagmentation is stopped with a Proteinase K solution. Tagmented DNA is bead purified and PCR amplified. |
| scATAC-seq56 | nanowell (SMARTer ICELL8, Takara Bio), single cell dispension | Cells are dispensed into wells of a 5,184 nanowell chip prior to tagmentation. Wells containing one living cell are selected for tagmentation and library preparation. |
| scATAC-seq57 | microfluidics chamber (Fluidigm) | Individual nuclei are tagmented and released DNA is PCR amplified in the integrated fluidic circuit (IFC). Amplified DNA is transferred to a 96-well plate for index PCR. |
| 10x scATAC-seq58 | droplet, 10x Genomics | Tagmentation without indexes. Tagmented DNA binds to barcoded beads in droplets. Initial amplification using linear amplification in droplets. Libraries are generated after pooling by PCR. |
| dsc/(dsci)ATAC-seq45 | (Indexing +) droplet (Bio-Rad) | Tagmentation with barcoded Tn5 to index at the molecular level. Tagmented nuclei are combined and encapsulated in droplets. Tagmented DNA binds to barcoded beads in droplets. The experiment can also be performed without indexing during tagmentation (dscATAC). |
| SNuBar-ATAC61 | indexing + droplet (10x Genomics) | Tagmentation with unindexed 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 and sample. Tagmented nuclei are combined and encapsulated in droplets. |
| HyDrop-ATAC62 | droplet, custom | Tagmentation is performed in bulk without indexes before loading onto a custom, open-source droplet-based platform. Hydrogel beads dissolve in droplets and release indexed primers that anneal to DNA fragments. Initial amplification using linear amplification. Libraries are generated after pooling by PCR. |
| sciATAC-seq60 | split-pool (2 rounds) | Nuclei are isolated and distributed into 96-well plates. Bulk nuclei in each well are tagmented using indexed Tn5. After pooling and dispensing/sorting of 15-25 nuclei-well into new plates a second barcode is introduced using indexed PCR primers. Custom sequencing recipe and primers. |
| snATAC-seq67 | split-pool (2 rounds) | Based on the original sciATAC strategy60. Here, Tn5 is loaded with a different set of transposons (universal T5; indexed T7) to omit custom sequencing recipes. Liquid handling robotics is used for pipetting steps in 96-well plates. After tagmentation, nuclei are pooled and 20 nuclei/well are sorted into new plates to introduce second barcode using indexed PCR primers (universal i7; indexed i5). Custom sequencing primers. |
| sci-ATAC-seq363 | split-pool (3 rounds) | Tagmentation without indexes. Barcoding is performed through ligation (two rounds) followed by index PCR. Additional round of indexing increases scale. Custom sequencing recipe and primers. |
| sciATAC-seq68 | split-pool (2 rounds) | Based on the original sciATAC strategy60. Fixed cells are distributed to 96-well plates and permeabilized. Indexed Tn5 is added to the wells for tagmentation. After tagmentation, wells are pooled, centrifuged, resuspended and distributed across a new set of 96-well plates. Each well is reverse crosslinked with a proteinase K buffer prior to PCR with indexed primers. |
| sciMAP-ATAC70 | split-pool (2 rounds) | Small biopsies are added to wells as starting material for indexed tagmentation. Custom sequencing recipe and primers. |
| scTHS-seq71 | split-pool (2 rounds) | In ATAC-seq only 50% of molecules have forward and reverse adapter and can be amplified by PCR. Here, tagmentation with T7 promoter sequence and in vitro transcription are used to overcome this limitation. |
| S3-ATAC72 | split-pool (2 rounds) | In ATAC-seq only 50% of molecules have forward and reverse adapter and can be amplified by PCR. Here, single indexed adapter Tn5 and adapter switching are used to overcome this limitation. Tagmentation is performed using Tn5 with a single indexed adapter containing a uracil in the transposon sequence. Nuclei are pooled after tagmentation and redistributed into new plates. A uracil intolerant polymerase is used for gap fill in. An oligo containing a 3’ lock nuclei acid (LNA) is annealed to the unindexed fragment end. Fragments are extended to copy the oliqo sequence and are finally amplified by PCR using indexed primers. |
| Histone modifications and/or DNA binding proteins | ||
| scChIP-seq78 | droplet, custom | Cells are encapsulated with micrococcal nuclease (MNase). These droplets are merged with droplets containing barcoded oligonucleotides and the adaptors are ligated to the nucleosomes. Indexed chromatin fragments from 100 cells are used as input for ChIP. Profiled modification: H3K4me3 |
| scChIP-seq79 | droplet, custom | MNase digestion is followed by single cell barcoding of nucleosomes and pull down of pooled nucleosomes. For barcoding, nucleosome-containing droplets are fused with droplets containing hydrogel beads carrying barcoded DNA adapters. Subsequently the barcoded DNA adapters are cleaved off the beads with UV light and ligated to the nucleosomes. Profiled modification: H3K27me3 |
| itChIP-seq80 | split-pool (1 round) | Fixed individual cells are sorted into 96-well plates and incubated with SDS to open the chromatin. The genomic DNA is fragmented with barcoded Tn5 and indexed soluble chromatin is used as input for ChIP-seq library preparation. Profiled modification: H3K27ac |
| scChIC-seq84 | plate-based, single cell dispension | Cells are incubated with MNase-Protein A and antibody or MNase-Antibody complexes. After incubation, single cells are sorted and MNase is activated. Profiled modifications: H3K4me3, H3K27me3 |
| uliCUT&RUN85 | tube/plate-based, single cell dispension | Cells are sorted prior to incubation with primary antibody followed by incubation with MNase-Protein A. Profiled factors: SOX2/NANOG |
| iscChIC-seq86 | split-pool (2 rounds) | Fixed cells are incubated with MNase-Protein A and antibody complexes. MNase is activated and cells distributed to 96-well plate where barcoded adapters are ligated. Wells are pooled and redistributed into new set of plates where PCR indexing is performed. Profiled modifications: H3K4me3, H3K27me3 |
| scCUT&Tag83 | nanowell (SMARTer ICELL8, Takara), single cell dispension | Cells are permeabilized to isolate nuclei. Nuclei are incubated with primary antibodies and washed several times. Nuclei are incubated with pA-Tn5 in high-salt buffer, washed and tagmented. After tagmentation in bulk, nuclei are dispensed on the SMARTer ICELL8 single-cell system and cells are indexed during PCR amplification. Profiled modifications: H3K4me2, H3K27me3 |
| scCUT&Tag89 | droplet, 10x Genomics | Adaptation of CUT&Tag83. After tagmentation in bulk, nuclei suspensions are encapsulated into gel emulsions on a Chromium controller and libraries are prepared following the 10x scATAC-seq protocol. Profiled modification: H3K27me3 |
| scCUT&Tag90 | droplet, 10x Genomics | Adaptation of CUT&Tag83. After tagmentation in bulk, nuclei suspensions are encapsulated into gel emulsions on a Chromium controller and libraries are prepared following the 10x scATAC-seq protocol. Profiled modifications/factors: H3K4me3, H3K27ac, H3K36me3, H3K27me3, RAD21, OLIG2 |
| CoBATCH88 | split-pool (2 rounds) | Permeabilized fixed or non-fixed cells are first incubated with antibodies. Cells are sorted into wells containing protein A (pA)-Tn5 with unique combinations of T5 and T7 barcodes. After tagmentation, cells are combined and redistributed into another set of 96-well plate for PCR barcoding. Profiled modification: H3K27ac |
| iACT-seq87 | split-pool (2 rounds) | Antibodies are first incubated with barcoded protein A (pA)-Tn5. These complexes are then added to permeabilized cells and incubated to bind to chromatin. Cells are combined, sorted into a 96-well plate and tagmentation is started. After tagmentation, DNA in each well is purified using phenol-chloroform extraction and transferred to new tubes for PCR barcoding. Profiled modification: H3K4me3 |
| sciTIP-seq91 | split-pool (2 rounds | Adaptation of CUT&Tag83, but using indexed transposons with T7 promoter sequence similar to scTHS-seq71 to overcome the limitation that only 50% of tagmented fragments can be PCR amplified. Cells are tagged with primary and secondary antibodies in bulk, then distributed to 96-well plates for indexed pA-Tn5 binding and tagmentation. After tagmentation, cells are pooled and redistributed to another plate for in vitro transcription followed by index PCR in the same well. Profiled modification/factors: H3K27me3, H3K27ac, H3K9me3, CTCF, RNA Pol II |
| Chromatin architecture | ||
| scHi-C95 | plate-based, single cell dispension | Crosslinking, restriction enzyme digestion, biotin fill-in and ligation are performed in bulk nuclei. Nuclei are selected under a microscope and libraries generated |
| scHi-C96 | plate-based, single cell dispension | This protocol combines imaging and scHiC. Cells are crosslinked in bulk. Nuclei are extracted and sorted into wells. Nuclei are imaged, overlaid with agarose and permeabilized. Restriction enzyme digestion, biotin fill-in and ligation are performed in individual wells. Libraries are generated either by adapter ligation or tagmentation. |
| snHi-C97 | plate-based, single cell dispension | Crosslinking, restriction enzyme digestion and ligation are performed in bulk or in individual nuclei. Biotin steps are omitted to increase fragment numbers. After ligation, whole genome amplification is performed prior to library preparation. |
| Dip-C98,99 | plate-based, single cell dispension | Crosslinking, restriction enzyme digestion and ligation are performed in bulk. Biotin-related steps are omitted, and an efficient transposon based whole-genome amplification with multiplex end-tagging amplification is performed. |
| scHi-C100 | plate-based, single cell dispension | Crosslinking, restriction enzyme digestion and ligation are performed in bulk. Nuclei are dispensed in a 96-well plate, tagmented with Tn5 and biotinylated fragments are bound to beads. Fragments are amplified from the beads with PCR using indexed primers. |
| sciHi-C101,102 | split-pool (2 rounds) | Crosslinked nuclei are distributed into 96-well plates after restriction enzyme digestion. The first barcode is introduced during biotinylated adapter ligation. After pooling and redistribution another barcode is introduced using another round of adapter ligation. |
| s3-GCC72 | split-pool (2 rounds) | Crosslinked nuclei are distributed into 96-well plates after restriction enzyme digestion and ligation. Biotin-related steps are omitted. Libraries are prepared using single indexed adapter Tn5 and adapter switching as in s3-ATAC-seq72. |
| scSPRITE103 | split-pool (3 rounds for nuclei, 3 rounds for chromatin clusters) | Crosslinking, restriction enzyme digestion is performed in bulk. Nuclei are distributed across a 96-well plate and fragmented DNA in each nucleus is tagged by ligation with a unique cell barcode through three rounds of split-pooling. A small subset of nuclei is sonicated to shear the chromatin. Crosslinked chromatin is bound to magnetic N-hydroxysuccinimide beads. Bead-bound chromatin complexes are barcoded for another three rounds to generate unique label for clusters in close spatial proximity. Detects multiway contacts. |
scBS-seq, single-cell bisulfite sequencing; scWGBS, single-cell whole genome bisulfite sequencing, snmC-seq, single nucleus methylcytosine sequencing; scRRBS, single-cell reduced representation bisulfite sequencing; XRBS, single-cell extended-representation bisulfite sequencing, sci-MET, single-cell combinatorial indexing for methylation analysis; scDNAse-seq, single-cell DNase sequencing; iscDNase-seq, indexing single-cell DNase sequencing; scATAC-seq, single-cell assay for transposase-accessible chromatin with high-throughput sequencing; dscATAC-seq, droplet single-cell assay for ATAC-seq; dsciATAC-seq, droplet-based single-cell combinatorial indexing for ATAC-seq; SNuBar-ATAC, single nucleus barcoding approach for ATAC-seq; HyDrop-ATAC, hydrogel-based droplet microfluidics for scATAC-seq; sciATAC-seq, single-cell combinatorial indexing ATAC-seq; snATAC-seq, single-nucleus ATAC-seq; sciMAP-ATAC, single-cell combinatorial indexing on microbiopsies assigned to positions for ATAC-seq; scTHS-seq, single-cell transposome hypersensitive sites sequencing; s3-ATAC, Drop-ChIP, Droplet-based single-cell chromatin immunoprecipitation sequencing, scChIP-seq, single-cell chromatin immunoprecipitation followed by sequencing; sc-itChIP-seq, single-cell indexing and tagmentation-based ChIP-seq; scChIC-seq, single-cell chromatin immunocleavage sequencing; uliCUT&RUN, ultra-low input cleavage under targets and release using nuclease; iscChIC-seq, indexing single-cell immunocleavage sequencing; scCUT&Tag, single-cell cleavage under targets and tagmentation; CoBATCH, combinatorial barcoding and targeted chromatin release; iACT-seq, indexing antibody-guided chromatin tagmentation sequencing; scTIP-seq, single-cell targeted insertion of promoters sequencing; scHi-C, single-cell HiC; Dip-C, diploid chromatin conformation capture; snHi-C, single-nucleus Hi-C, single-cell combinatorial indexing Hi-C; s3-GCC, symmetrical strand single-cell combinatorial indexing genome conformation capture; scSPRITE, single-cell split-pool recognition of interactions by tag extension;