The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.
Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses.
In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell.
Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.
Single-cell technology is entering the era of single-cell multiomics, which integrates individual single-cell omics technologies1. Recently, single-cell transcriptomics has been combined with methods for detecting chromatin accessibility (scNMT-seq2 and SHARE-seq3) or histone modifications (Paired-seq4 and Paired-Tag5). More recently, single-cell transcriptomics and proteomics were integrated with chromatin accessibility (DOGMA-seq6). These methods use transposase-based tagging for detecting chromatin accessibility or histone modifications.
Transposase-based approaches cleave genomic DNA and add a DNA barcode at the end of the genomic DNA fragment. Each cleaved genomic fragment can only accept up to two DNA barcodes (= one epigenetic mark per cleavage site), and the genomic DNA at the cleavage site is lost. Therefore, cleavage-based approaches have a trade-off between the number of epigenetic marks tested and the signal density. This hampers the analysis of multiple epigenetic marks in the same single cell. A single-cell epigenomic method that does not cleave the genomic DNA was developed to overcome this issue7,8.
In addition to the cleavage-derived issue mentioned above, transposase-based approaches have other limitations. In single-cell epigenome analysis, it is critical to know the location of histones and DNA-associated proteins on the genome. In current approaches, this is accomplished by using unfixed single cells and retention of protein-DNA and protein-protein interactions. However, this generates strong bias to accessible chromatin regions, even in the analysis of histone modifications 9. The location of histones and genome-associated proteins on the genome can be preserved without crosslinking protein-DNA and protein-protein, using a polyacrylamide scaffold7,8. This approach reduces the structural bias observed in current approaches that depend upon protein-DNA and protein-protein interactions.
Transposase-based approaches can acquire signals only once from a single cell. Therefore, it is difficult to delineate the complete epigenome of a single cell due to the drop-off of the signals. Reusable single cells have been developed to overcome current limitations by allowing iterative epigenomic analysis in the same single cell.
NOTE: A schematic representation of the method is shown in Figure 1.
Figure 1: Schematic representation of the protocol workflow. Steps 7.2-13 are explained through schematic representations. Each row indicates a step in the protocol. A cellular protein colored in green is a human nucleosome generated based on a crystal structure (PDB: 6M4G). Please click here to view a larger version of this figure.
1. Equilibration of desalting columns
NOTE: Desalting spin columns are equilibrated as described in the following steps. The equilibrated desalting columns are used in steps 2.1, 3.4, and 4.6.
2. Buffer exchange of antibodies
NOTE: Remove glycerol, arginine, and sodium azide from anti-H3K27ac10, anti-H3K27me310, anti-Med111, and anti-Pol II10 (see buffer composition shown in Table 1). All following procedures are performed under a clean hood to avoid DNase contamination. Time: 1 h
3. Antibody activation
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 2.5 h
4. Activation of DNA probe
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 2.5 h
5. Conjugation of S-HyNic-modified antibody and S-4FB-modified Antibody Probe
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 2 h
6. Preparation of core magnetic beads
NOTE: In this method, a single cell is embedded into a bilayered acrylamide bead (see Figure 2). The core is a magnetic polyacrylamide bead. The outer layer is polyacrylamide alone. The core magnetic beads are generated in this section. This section is not essential for the experiment. Time: 3 h
Figure 2: Structure of a bilayered polyacrylamide bead for visibility and easy handling in REpi-seq experiments. (A) Magnetic nanoparticles from step 6.6 after centrifugation. The magnetic nanoparticles are modified with monomeric acrylamide and integrated into the polyacrylamide magnetic bead shown in B.(B) Schematic representation of a reusable single cell with a polyacrylamide magnetic bead. Please click here to view a larger version of this figure.
7. Modifying the amino group of cellular proteins with monomer acrylamide
NOTE: REpi-seq was designed to analyze the epigenome of mouse and human cells at the single-cell level. Each step must be optimized when using this method on cells of species other than mouse or human.
8. Preparation of reusable single cells
Figure 3: Automatic single-cell picking and transfer into a 96-well PCR plate in step 8.2. (A) Overview of a single cell-picking system. A single cell-picking robot is in a laminar flow clean hood to avoid contamination. (B) A 24-well plate with 4 nL nanowells inside the well. (C) Cell distribution in a well from the 24-well plate. Green dots are cells identified as a single cell in each 4 nL nanowell. Magenta dots are cells identified as doublets or multiplets of cells. (D) Brightfield image of the well in the 24-well plate. A green square is a 4 nL nanowell containing a single cell. A magenta square is a 4 nL nanowell containing multiple cells. (E) Magnified field of some 4 nL nanowells. Bright dots are single cells in 4 nL nanowells. The single cell-picking system identifies nanowells containing a single cell by acquiring brightfield and fluorescence images of cells with DAPI staining. Identified single cells are transferred from the 4 nL nanowell to a well of a 96-well PCR plate. Scale bars = 2 mm (C, D), 100 µm (E). Abbreviation = DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 4: Generating reusable single cells using a liquid-handling robot. (A) A deck of the liquid-handling robot. The deck has 11 slots for pipette tip racks (P300 tip: Slots 1-3, P20 tip: Slots 5-6), 2 mL deep-well 96-well plate (Slot 4), two 96-well PCR plates containing a single cell per well (Slots 7 and 10), and two flat-bottom 96-well plates for liquid waste (Slots 8 and 11). (B) The deck after placing the labware. (C) Schematic representation of robotic pipetting in step 8.8.1. The program removes the supernatant without aspirating a single cell from the bottom of the 96-well PCR plate. (D) Reusable single cells generated using the Supplemental Code 1. Please click here to view a larger version of this figure.
9. Random primer annealing and extension
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Asterisks (*) at the following steps indicate that a magnetic separator can be used to control the position of the polyacrylamide beads containing a reusable single cell in the tube. However, the use of the magnetic separator is not essential. By moving down the pipette tip slowly along the wall of the tube, the beads are pushed up for washing or buffer exchange. Time: 9 h
10. Antibody binding
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 1.5 h
11. Proximity ligation of antibody probe and proximally extended random primer
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Asterisks (*) in the following steps indicate where a magnetic separator can be used to control the position of polyacrylamide beads containing a reusable single cell in the tube. However, the use of the magnetic separator is not essential. By moving down the pipette tip slowly along the wall of the tube, the beads can be pushed up for washing or buffer exchange. Time: 6 h
12. Full extension of the 1st random primer
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 4.5 h
13. Multiple displacement amplification
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 2.5 h (steps 13.1-13.2) + 15 min (steps 13.3-13.4) + 1 day (steps 13.5-13.10)
14. Phenol-chloroform purification and polyethylene glycol precipitation
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 1.5 h
15. In vitro transcription
NOTE: The following procedure is performed under a clean hood to avoid DNase and RNase contamination. Time: 5 h
16. RNA purification
NOTE: The following procedure is performed under a clean hood to avoid DNase and RNase contamination. Time: 2 h
17. Reverse transcription
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 1 h
18. Second strand synthesis
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 2.5 h
19. Restriction enzyme digestion and size selection
NOTE: The following procedure is performed under a clean hood to avoid DNase contamination. Time: 3 h (steps 19.1-19.7)
K562 single cells were generated using the protocol described in step 8 (see Figure 5). Single cells were embedded in the outer layer of the polyacrylamide bead. Cell DNA was stained and visualized using an intercalator dye for DNA staining.
Figure 5: Generated reusable single cells. Cells are stained with an intercalator dye for DNA (green fluorescence). White arrows indicate single cells embedded in the outer layer of polyacrylamide beads. The reusable single cell is placed in a 96-well flat-bottom plate. Images were taken by a scanning microscope, BZ-X710. Scale bar = 1 mm. Please click here to view a larger version of this figure.
The results shown in Figure 6 supported the generation of the desired DNA products. DNA products from step 19.7 were cleaved by the BciVI restriction enzyme into 19-20 bp, 31-32 bp, 49 bp, and >49 bp fragments. These results supported the conclusion that most products contain Antibody Probe-derived sequences and 2nd random primer-derived sequences. The DNA products were subsequently ligated with the Illumina adapter using a TruSeq Nano kit and sequenced using an Illumina NovaSeq6000 sequencer.
Figure 6: DNA products before and after BciVI restriction enzyme digestion. (A) BciVI digestion generated the expected 19-20 bp fragments, 31-32 bp fragments, and the desired products (>49 bp) containing Antibody Barcode, Ligated sequence, genomic DNA, and Cell barcode. (B) Size distribution of the final products at the end of step 19.7. Constructed DNA library was analyzed by capillary electrophoresis. The light pink line indicates the desired products containing sequencing adaptors, antibody barcode, and cell barcode. Please click here to view a larger version of this figure.
The sequencing results indicated that the products generated by following steps 7-19 contain the Antibody Probe, ligated sequence, genome sequence, and cell barcode. Unique mapped reads from anti-H3K27ac and anti-H3K27me3 were significantly more numerous than from control IgG (see Figure 7A). This indicates that the specific binding of anti-H3K27ac and anti-H3K27me3 increases the number of the desired products. The most advanced technology for single-cell epigenome analysis, Paired-Tag, acquires around 2,000 unique reads of H3K27ac and 1,500 unique reads of H3K27me3 per nucleus. Our results showed a much higher number of unique reads (average 699,398 H3K27ac signals/cell and 505,433 H3K27me3 signals/cell). The results also supported the conclusion that repeated experiments using the same reusable single cell reduce loss of signal and increase signal density.
The REpi-seq signals shown in Figure 7A were evaluated by comparing the REpi-seq data with bulk ChIP-seq data. In Figure 7B, on average, 91% ± 3.24% of H3K27ac signals from REpi-seq overlapped with H3K27ac peaks in bulk ChIP-seq. This analysis measures the level of "precision" in single-cell epigenome analysis18. The precision of current single-cell epigenomic methods for the active histone mark H3K4me3 is 53% (Drop-ChIP; H3K4me3)18, 50% (scChIC-seq; H3K4me3)19, and 60% (ACT-seq; H3K4me3)20. In addition, the precision of REpi-seq for the inactive histone mark H3K27me3 was, on average, 52.09% ± 3.71% (Figure 7C), whereas the precision for H3K27me3 was 47% in single-cell ChIC-seq19. In conclusion, REpi-seq displays high precision in detecting H3K27ac and H3K27me3.
We also analyzed the "sensitivity"18 of REpi-seq, which measures how many peaks of bulk ChIP-seq were detected by REpi-seq reproduced reads (Figure 7D,E). The sensitivity of REpi-seq was 55.30% in H3K27ac and 50.94% in H3K27me3. In other single-cell epigenome methods, the sensitivity is 5% in Drop-ChIP (H3K4me3)18, 5% in ACT-seq (H3K4me3)20, and 9.5% in scChIC-seq (H3K27me3)19. These results indicate that REpi-seq is sensitive at detecting epigenetic signals.
Figure 7: Signal numbers, precision, and sensitivity in REpi-seq. The same experiments were repeated 3 times with the same reusable single cell (a K562 cell). (A) Acquired signals from the same eight single cells. Each dot represents a reusable single cell. Unique signals from control IgG (black), anti-H3K27me3 (blue), and anti-H3K27ac (red) in the same single cells were counted. Anti-H3K27me3 and anti-H3K27ac signals were significantly higher than control IgG, indicating that specific binding of antibodies generates signals more efficiently than control IgG. Bar: mean, error bar: standard deviation. (B, C) Precision of REpi-seq H3K27ac (B) and H3K27me3 (C) unique reads. The precision of unique reads derived from REpi-seq was based on overlapping with known ChIP-seq peaks from K562 bulk cell analysis (H3K27ac: SRR3144862; H3K27me3: SRR069083). The percentages of REpi-seq H3K27ac and H3K27me3 reads confirmed by ChIP-seq peaks were calculated in each single cell. The results are expressed as the average percent of 8 single cells. (D, E) Sensitivity of REpi-seq at detecting H3K27ac (D) and H3K27me3 (E) known peaks. Unique reads of REpi-seq were used. H3K27ac and H3K27me3 peaks identified by ChIP-seq of K562 bulk cells in the ECODE Project (H3K27ac: ENCFF038DDS; H3K27me3: ENCFF031FSF) were recognized by REpi-seq unique reads. The results are expressed as the average percent ChIP-seq peaks recognized by REpi-seq unique reads. Panels B–E were reproduced from Ohnuki et al.7. Please click here to view a larger version of this figure.
Table 1: Buffers used in this protocol. Please click here to download this Table.
Table 2: Antibodies and IgG control used in this protocol. Please click here to download this Table.
Table 3: Oligonucleotide DNA used in this protocol. Please click here to download this Table.
Supplemental Video S1: Automated single-cell picking and transfer into a 96-well PCR plate (step 8.2.4). Please click here to download this Video.
Supplemental Video S2: Supernatant removal from a well containing a single cell using a liquid handling robot (step 8.2.8.1). Please click here to download this Video.
Supplemental Video S3: Adding 4% TEMED/mineral oil to a well containing a single cell using a liquid handling robot (step 8.2.8.2). Please click here to download this Video.
Supplemental Code 1: Automated program of the liquid handling robot from steps 8.2.8.1-8.2.8.2. Please click here to download this File.
This article describes the step-by-step protocol for the recently reported single-cell multiepigenomic analysis using reusable single cells7. In the subsequent paragraphs, we discuss critical points, emphasizing potential limitations in the protocol.
One of the critical points throughout the protocol (from steps 7.2-13) is avoiding DNase contamination. A single cell only has two copies of genomic DNA. Therefore, damaging genomic DNA critically reduces the signal number. Avoiding contamination with DNase is essential to protect the integrity of genomic DNA.
Permeability of the reagents across the cell membrane/cell wall is also critical throughout the protocol. REpi-seq was designed to analyze the epigenome of mouse and human cells at the single-cell level. The optimal conditions for this experimental method are expected to vary depending on the permeability of cell types and nuclear membrane to each reagent. Therefore, when the method is applied to cells other than mouse and human cells, such as plant cells, yeast, and bacteria, the conditions at each step need optimization.
We believe that a critical point specific to step 9.5 (annealing the first random primer) is the rapid speed of cooling after denaturing the genomic DNA. Slow and gradual cooling after denaturing may reduce the annealing efficiency of the random primers binding to the genomic DNA. Moreover, in step 11 (proximity ligation), a critical point is the buffer composition. Buffers containing polyethylene glycol (PEG) are widely used for faster ligation methods in molecular biology applications. A low concentration of PEG (e.g., <7.5%) promotes the quick formation of double-stranded DNA without DNA precipitation. However, we observed that the ligation buffer containing PEG induces shrinkage of the single-cell gel bead containing the single cell (reusable single cell), suggesting that the mesh size of the polyacrylamide scaffold had shrunk. As this shrinkage may cause reduced accessibility of T4 DNA ligase, we decided not to use the faster ligation protocol for REpi-seq.
In REpi-seq, the results can be evaluated by the redetection frequency of signals in the same single cells. The redetection frequency is useful to monitor reactions, including proximity ligation with the antibody probe and first random primer. We previously reported7 that 62.03% of H3K27ac reads in one experiment was confirmed in two replicate experiments, suggesting that the efficiency of proximity ligation in individual experiments is higher than the overall redetection percentage in repeated experiments.
We have also previously reported7 the successful detection of RNA polymerase II (Pol II) binding to DNA in reusable single cells. Pol II binding is difficult to capture with the current single-cell epigenome methods because of the temporal and unstable nature of this binding.
Current transposase-based CUT&Tag approaches for single-cell epigenome analysis require the preservation of nucleosome-DNA interaction. A specific antibody binds to a histone modification; then, transposase attached to the antibody cleaves the genomic DNA and inserts a DNA barcode at the DNA cleavage site. This reaction depends on nucleosome (histone)-DNA interaction. Therefore, it is essential that nucleosome-DNA interactions and the structure of cellular proteins and chromatin remain intact. Hence, CUT&Tag approaches are inevitably biased to accessible regions of chromatin structure in the analysis of histone modifications.
The inevitable bias toward the accessible chromatin regions hampers the augmentation of the number of epigenetic signals in single-cell epigenome analysis. Chromatin structures are formed by multiple types of molecular interactions, including DNA-nucleosome and nucleosome-nucleosome interactions through nucleosome-associated proteins. Hence, we opted not to preserve DNA-protein and protein-protein interactions in the reusable single cells while preserving the location of cellular proteins. This feature is accomplished with the use of monomeric acrylamide and a paraformaldehyde mixture (protocol step 7), which modifies the amino group on cellular proteins rather than crosslinking protein to protein or protein to DNA.
The location of histones and genome-associated proteins is retained by the connection to the polyacrylamide polymer. Nucleosomes are denatured around 71-74 °C21. Therefore, in the reusable single cell, histones are likely denatured and relaxed/dissociated from each other after the annealing step of the 1st random primer (94 °C). This may relax the heterochromatin structure and improve the accessibility of antibodies and other molecules to heterochromatin regions. This conclusion is supported by our reported results7 showing that bias associated with chromatin structure is reduced in reusable single cells compared to CUT&Tag approaches9.
The connection of histone H3 to polyacrylamide is maintained after at least 3 rounds of heat denaturing as the location of histones in the reusable single cell is preserved over repeated experiments7. This feature allows repetition of the same experiment using the same single cell. Repeated experiments contribute to increasing the number of signals from a single cell compared to CUT&Tag approaches.
Verifiability is a fundamental principle in science. However, verifying experimental results of a single cell is not feasible in CUT&Tag approaches, as repeated experiments with the same cells are not possible. The genomic DNA is cleaved by transposase and receives the DNA barcode for one epigenetic mark. The cleaved genomic DNA is released from its original location. Therefore, the CUT&Tag approach is at a disadvantage for the analysis of multiple epigenetic marks in the same single cell. This feature of CUT&Tag methods underlies the trade-off of decreased signal density with an increased number of epigenetic marks per single cell.
To avoid this issue, we copied genomic DNA rather than cutting the genome and then tagged the copied genomic DNA with an antibody DNA probe. REpi-seq allows the analysis of the same single cells and increases signal density. It also provides a means for verifiability of experimental results, multiplexing epigenomic analysis, and solving the trade-off issue in CUT&Tag approaches. Although the method overcomes the limitations of transposase-based epigenomic analysis, it is not yet capable of analyzing large numbers of cells at a single-cell level. Further development is needed.
In conclusion, REpi-seq copies genomic DNA rather than cutting the genome and then tags the copied genomic DNA with an antibody DNA probe. REpi-seq is still in infancy and needs to increase throughputs of cells. However, REpi-seq has strengths, which overcomes the limitations in current single-cell epigenome methods: 1) increasing signal density by reducing drop-outs of signals through repeated experiments in the same single cells; 2) reducing structural bias by reducing inaccessible regions based on chromatin structure; 3) providing verifiability of experimental results by repeated experiments in the same single cell, 4) signal identification based on redetection probability of the same signal in every single cell; 5) analyzing multiple epigenetic marks in the same single cell without competition among epigenetic marks. These advances allow analyzing epigenetic mechanisms in a single cell, which is an important application and not feasible with the other single-cell epigenomic methods.
The authors have nothing to disclose.
We thank Drs. David Sanchez-Martin and Christopher B. Buck for comments during the conceptualization stage of the project. We also thank the Genomics Core, Center for Cancer Research, National Cancer Institute, National Institutes of Health for help in preliminary experiments, and the Collaborative Bioinformatics Resource, CCR, NCI, NIH for advice in computational analysis. We thank Ms. Anna Word for helping with the optimization of DNA polymerases used in the method. This work utilized the computational resources of the NIHHPC Biowulf cluster (http://hpc.nih.gov). This project is supported by the Intramural Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health, the NCI Director's Innovation Award (#397172), and Federal funds from the National Cancer Institute under Contract No. HHSN261200800001E. We thank Drs. Tom Misteli, Carol Thiele, Douglas R. Lowy, and all members of Laboratory of Cellular Oncology for productive comments.
10x CutSmart buffer | New England BioLabs | B6004 | 10x Digestion buffer |
200 proof ethanol | Warner-Graham Company | 200 proof | Ethanol |
5-Hydroxymethylcytosine (5-hmC) Monoclonal Antibody [HMC/4D9] | Epigentek | A-1018-100 | Anti-5hmC |
Acridine Orange/Propidium Iodide Stain | Logos Biosystems | F23001 | Cell counter |
Acrylamide solution, 40% in H2O, for molecular biology | MilliporeSigma | 01697-500ML | 40% acrylamide solution |
All-in-One Fluorescence Microscope BZ-X710 | Keyence | BZ-X710 | Scanning microscope |
Amicon Ultra-0.5 Centrifugal Filter Unit | MilliporeSigma | UFC510024 | Ultrafiltration cassette |
Ammonium persulfate for molecular biology | MilliporeSigma | A3678-100G | Ammonium persulfate powder |
Anhydrous DMF | Vector laboratories | S-4001-005 | Anhydrous N,N-dimethylformamide (DMF) |
Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] | Abcam | ab5408 | Anti-Pol II |
Anti-TRAP220/MED1 (phospho T1457) antibody | Abcam | ab60950 | Anti-Med1 |
BciVI | New England BioLabs | R0596L | BciVI |
Bovine Serum Albumin solution, 20 mg/mL in H2O, low bioburden, protease-free, for molecular biology | MilliporeSigma | B8667-5ML | 20% BSA (Table 7) |
Bst DNA Polymerase, Large Fragment | New England BioLabs | M0275L | Bst DNA polymerase |
BT10 Series 10 µl Barrier Tip | NEPTUNE | BT10 | P10 low-retention tip |
CellCelector | Automated Lab Solutions | N/A | Automated single cell picking robot |
CellCelector 4 nl nanowell plates for single cell cloning, Plate S200-100 100K, 24 well,ULA | Automated Lab Solutions | CC0079 | 4 nL nanowell plate |
Chloroform | MilliporeSigma | Chloroform | |
Corning Costar 96-Well, Cell Culture-Treated, Flat-Bottom Microplate | Corning | 3596 | Flat-bottom 96-well plates |
Deep Vent (exo-) DNA Polymerase | New England BioLabs | M0259L | Exo– DNA polymerase |
DNA LoBind Tubes, 0.5 mL | Eppendorf | 30108035 | 0.5 mL DNA low-binding tube |
DNA Oligo, 1st random primer | Integrated DNA Technologies | N/A, see Table 3 | 1st random primer |
DNA Oligo, 2nd random primer Cell#01 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#02 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#03 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#04 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#05 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#06 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#07 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#08 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#09 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#10 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#11 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd random primer Cell#12 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
DNA Oligo, 2nd synthesis primer | Integrated DNA Technologies | N/A, see Table 3 | 2nd synthesis primer |
DNA Oligo, Ligation Adaptor | Integrated DNA Technologies | N/A, see Table 3 | Ligation Adaptor |
DNA Oligo, Reverse Transcription primer | Integrated DNA Technologies | N/A, see Table 3 | Reverse Transcription primer |
DNase I (RNase-free) | New England BioLabs | M0303L | DNase I (RNase-free, 4 U). |
DNase I Reaction Buffer | New England BioLabs | B0303S | 10x DNase I buffer (NEB) |
dNTP Mix (10 mM each) | Thermo Fisher | R0192 | 10 mM dNTPs |
Fetal Bovine Serum, USA origin, Heat-inactivated | MilliporeSigma | F4135-500ML | Fetal bovine serum |
HiScribe T7 High Yield RNA Synthesis Kit | New England BioLabs | E2040S | In-vitro-transcription master mix |
Histone H3K27ac antibody | Active motif | 39133 | Anti-H3K27ac |
Histone H3K27me3 antibody | Active motif | 39155 | Anti-H3K27me3 |
IgG from rabbit serum | Millipore Sigma | I5006-10MG | Control IgG |
Iron oxide(II,III) magnetic nanopowder, 30 nm avg. part. size (TEM), NHS ester functionalized | MilliporeSigma | 747467-1G | NHS ester functionalized 30 nm iron oxide powder |
K-562 | American Type Culture Collection (ATCC) | CCL-243 | cells |
Linear Acrylamide (5 mg/mL) | Thermo Fisher | AM9520 | Linear Acrylamide |
LUNA-FL Dual Fluorescence Cell Counter | Logos Biosystems | L20001 | Cell counter |
LUNA Cell Counting Slides, 50 Slides | Logos Biosystems | L12001 | Cell counter |
Mineral oil, BioReagent, for molecular biology, light oil | MilliporeSigma | M5904-500ML | Mineral oil |
N,N,N′,N′-Tetramethylethylenediamine for molecular biology | MilliporeSigma | T7024-100ML | N,N,N′,N′-Tetramethylethylenediamine |
NaCl (5 M), RNase-free | Thermo Fisher | AM9760G | 5M NaCl |
NanoDrop Lite | Thermo Fisher | 2516 | Microvolume spectrophotometer |
NEST 2 mL 96-Well Deep Well Plate, V Bottom | Opentrons | N/A | 2 mL deep well 96-well plate |
Non-skirted 96-well PCR plate | Genesee Scientific | 27-405 | 96-well PCR plate |
NuSive GTG Agarose | Lonza | 50081 | Agarose |
OmniPur Acrylamide: Bis-acrylamide 19:1, 40% Solution | MilliporeSigma | 1300-500ML | 40%Acrylamide/Bis-acrylamide |
OT-2 lab robot | Opentrons | OT2 | Automated liquid handling robot |
Paraformaldehyde, EM Grade, Purified, 20% Aqueous Solution | Electron Microscopy Sciences | 15713 | 20% Pararmaldehyde |
PBS (10x), pH 7.4 | Thermo Fisher | 70011044 | 10x PBS |
PIPETMAN Classic P1000 | GILSON | F123602 | A P1000 pipette |
Protein LoBind Tubes, 1.5 mL | Eppendorf | 925000090 | 1.5 mL Protein low-binding tube |
QIAgen Gel Extraction kit | Qiagen | 28706 | A P1000 pipette |
Quant-iT PicoGreen dsDNA Assay | Thermo Fisher | P11495 | dsDNA specific intercalator dye |
Quick Ligation kit | New England BioLabs | M2200L | T4 DNA ligase (NEB) |
RNaseOUT Recombinant Ribonuclease Inhibitor | Thermo Fisher | 10777019 | RNAse inhibitor |
S-4FB Crosslinker (DMF-soluble) | Vector laboratories | S-1004-105 | Succinimidyl 4-formylbenzoate (S-4FB) |
S-HyNic | Vector laboratories | S-1002-105 | Succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic) |
Sodium Acetate, 3 M, pH 5.2, Molecular Biology Grade | MilliporeSigma | 567422-100ML | 3M Sodium acetate (pH 5.2) |
Sodium bicarbonate, 1M buffer soln., pH 8.5 | Alfa Aesar | J60408 | 1M sodium bicarbonate buffer, pH 8.5 |
Sodium phosphate dibasic for molecular biology | MilliporeSigma | S3264-250G | Na2HPO4 |
Sodium phosphate monobasic for molecular biology | MilliporeSigma | S3139-250G | NaH2PO4 |
SuperScript IV reverse transcriptase | Thermo Fisher | 18090050 | Reverse transcriptase |
SYBR Gold Nucleic Acid Gel Stain (10,000x Concentrate in DMSO) | Thermo Fisher | S11494 | An intercalator dye for DNA |
T4 DNA Ligase Reaction Buffer | New England BioLabs | B0202S | 10x T4 DNA ligase reaction buffer |
ThermoPol Reaction Buffer Pack | New England BioLabs | B9004S | 10x TPM-T buffer (Tris-HCl/Pottasium chloride/Magnesium sulfate/Triton X-100) |
TRIzol LS reagent | Thermo Fisher | 10296-028 | Guanidinium thiocyanate-phenol-chloroform extraction |
TruSeq Nano DNA library prep kit | Illumina | 20015965 | A DNA library preparation kit (see also the manufacturer's instruction) |
Ultramer DNA Oligo, Anti-5hmC_Ab#005 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
Ultramer DNA Oligo, Anti-H3K27ac_Ab#002 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
Ultramer DNA Oligo, Anti-H3K27me3_Ab#003 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
Ultramer DNA Oligo, Anti-Med1_Ab#004 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
Ultramer DNA Oligo, Anti-Pol II_Ab#006 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
Ultramer DNA Oligo, Control IgG_Ab#001 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for control IgG |
UltraPure 0.5 M EDTA, pH 8.0 | Thermo Fisher | 15575020 | 0.5M EDTA, pH 8.0 |
UltraPure DNase/RNase-Free Distilled Water | Thermo Fisher | 10977023 | Ultrapure water |
Zeba Splin Desalting Columns, 7K MWCO, 0.5 mL | Thermo Fisher | 89882 | Desalting column |