Double M.CviPI/eM.SssI and M.CviPI/BGT labeling was performed by incubating 50 ng of model DNA fragments with 4 U M.CviPI (New England Biolabs, NEB) and 2-fold molar excess of eM.SssI (TS) over CG targets (for M.CviPI/eM.SssI labeling) or 2.5 U BGT (TS) (for M.CviPI/BGT labeling), and 600 μM Ado-6-azide and 150 μM UDP-glc-azide (Jena Bioscience) cofactors, respectively, in a buffer (10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1 mg/ml BSA, 10 mM DTT). For the triple M.CviPI/eM.SssI/BGT labeling reactions, 50 ng of model DNA fragments were incubated with 4 U M.CviPI, 2-fold molar excess of eM.SssI over CG targets, 2.5 U BGT, 600 μM Ado-6-alkyne and 150 μM UDP-glc-azide in buffer (10 mM Tris-HCl (pH 8.5), 50 mM NaCl, 0.1 mg/ml BSA, 10 mM DTT). Reactions were incubated at 37°C for 1 h, followed by enzyme inactivation at 65°C for 20 min. Then, DNA was treated with 0.2 mg/ml Proteinase K (ProtK, TS) and 0.1% of SDS for 1 h at 50°C, and then for 20 min at 65°C. DNA was purified with a DNA clean and concentrator kit (Zymo research, ZR).
M cvipi
Manufactured by New England Biolabs
M.CviPI is a Type II DNA methyltransferase enzyme from the Chlorella virus. It recognizes and methylates the cytosine residue in the DNA sequence 5'-TGCGCA-3'.
Automatically generated - may contain errors
Lab products found in correlation
M cvipi reaction buffer, by New England Biolabs (4 mentions)
M sssi, by New England Biolabs (4 mentions)
Igepal ca 630, by Merck Group (3 mentions)
Rnasein, by Promega (2 mentions)
Rlt plus buffer, by Qiagen (2 mentions)
Gm12878 lymphoblast, by Coriell Institute for Medical Research (2 mentions)
G tube, by Covaris (2 mentions)
S adenosylmethionine sam, by New England Biolabs (2 mentions)
Proteinase k, by New England Biolabs (2 mentions)
Rnase inhibitor, by Takara Bio (1 mentions)
Proteinase inhibitor, by Merck Group (1 mentions)
Gc buffer, by New England Biolabs (1 mentions)
Novaseq s4, by Novogene (1 mentions)
Nebnext enzymatic methyl seq kit, by New England Biolabs (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
S adenosylmethionine, by New England Biolabs (1 mentions)
Protease, by Qiagen (1 mentions)
Dam dcm lambda phage gdna, by Thermo Fisher Scientific (1 mentions)
M cvipi, by Jena Biosciences (1 mentions)
Dna clean and concentrator kit, by Zymo Research (1 mentions)
14 protocols using m cvipi
1
Multicolor DNA Labeling Techniques
2
Simultaneous NOMe-seq and dSMF Protocol
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NOMe-seq/dSMF experiments were carried out as previously described [41 (link)], with some modifications. Cells were pelleted at 10,000 g, then crosslinked as described for ATAC-seq at 1% formaldehyde concentration.
Fixed cells were resuspended in 100 μL M.CviPI Reaction Buffer (50 mM Tris–HCl pH 8.5, 50 mM NaCl, 10 mM DTT), then treated with M.CviPI by adding 200 U of M.CviPI (NEB), SAM at 0.6 mM and sucrose at 300 mM, and incubating at 30 °C for 20 min. After this incubation, 128 pmol SAM and another 100 U of enzyme were added, and a further incubation at 30 °C for 20 min was carried out. For dSMF experiments, M.SssI treatment followed immediately, by adding 60 U of M.SssI (NEB), 128 pmol SAM, and MgCl2 at 10 mM and incubation at 30 °C for 20 min. The reaction was stopped by adding an equal volume of Stop Buffer (20 mM Tris–HCl pH 8.5, 600 mM NaCl, 1% SDS, 10 mM EDTA).
Crosslinks were reversed overnight at 65 °C, and DNA was isolated using the MinElute PCR Purification Kit (Qiagen, Cat # 28,006).
Enzymatically labeled DNA was then sheared on a Covaris E220, and converted into sequencing libraries following the EM-seq protocol, using the NEBNext Enzymatic Methyl-seq Kit (NEB, Cat # E7120L).
SMF/NOMe-seq libraries were sequenced as 2 × 150mers on a NovaSeq S4 through Novogene to a depth of ∼100 × for 200-bp fragments.
Fixed cells were resuspended in 100 μL M.CviPI Reaction Buffer (50 mM Tris–HCl pH 8.5, 50 mM NaCl, 10 mM DTT), then treated with M.CviPI by adding 200 U of M.CviPI (NEB), SAM at 0.6 mM and sucrose at 300 mM, and incubating at 30 °C for 20 min. After this incubation, 128 pmol SAM and another 100 U of enzyme were added, and a further incubation at 30 °C for 20 min was carried out. For dSMF experiments, M.SssI treatment followed immediately, by adding 60 U of M.SssI (NEB), 128 pmol SAM, and MgCl2 at 10 mM and incubation at 30 °C for 20 min. The reaction was stopped by adding an equal volume of Stop Buffer (20 mM Tris–HCl pH 8.5, 600 mM NaCl, 1% SDS, 10 mM EDTA).
Crosslinks were reversed overnight at 65 °C, and DNA was isolated using the MinElute PCR Purification Kit (Qiagen, Cat # 28,006).
Enzymatically labeled DNA was then sheared on a Covaris E220, and converted into sequencing libraries following the EM-seq protocol, using the NEBNext Enzymatic Methyl-seq Kit (NEB, Cat # E7120L).
SMF/NOMe-seq libraries were sequenced as 2 × 150mers on a NovaSeq S4 through Novogene to a depth of ∼100 × for 200-bp fragments.
3
Single-cell Bisulfite Sequencing Protocol
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We first prepared a cell lysate, methylated the chromatin in vitro, released genomic DNA from the chromatin, and treated the genomic DNA with bisulfite in a one-tube reaction to avoid loss of material in the isolation and purification steps. Next, the bisulfite-treated genomic DNA from a single cell was amplified by a PBAT strategy to obtain sufficient material for sequencing. Briefly, a single cell or blastomere was picked using a mouth pipette and transferred into a 0.25 ml PCR tube containing 3.5 μl of ice-cold lysate buffer (50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 10 mM dithiothreitol, 0.25 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride and 0.5% NP-40, plus 1 pg λDNA) and was kept on ice for 10 min. Then, the GpC methyltransferase M.CviPI and S-adenosylmethionine (New England Biolabs, M0227L) were added to the lysate to a final volume of 5 μl containing 1 U/μl M.CviPI and 160 μM SAM. In vitro methylation of single-cell nuclei was performed by incubating the mixture in a thermocycler at 37 °C for 30 min before heat inactivation for 20 min at 65 °C. After in vitro methylation, 0.5 μl of 20 mg/ml protease (Qiagen) was added and the mixture was incubated for 3 h at 50 °C to release genomic DNA. The released genomic DNA was then bisulfite converted and used to prepare the single-cell COOL-seq Library.
4
Single-cell RNA/DNA methylation assay
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After collecting suspensions of EpCAM-positive cells isolated by MACS, we prepared a 2.5 µL cell lysis and in vitro methylation (IVM) mixture containing 4 U RNase inhibitor (Takara, Cat# 2313B), 0.25% IGEPAL CA-630 (Sigma, Cat# I3021), 1× GC reaction buffer and 2 U M.CviPI (NEB, Cat# M0227L). An individual cell was placed into the 2.5 µL IVM mixture and then gently vortexed for 10 s before running the IVM reaction on the thermocycler at 37 °C for 20 min and then at 65 °C for 25 min. Then, the cell lysate could be directly processed for RNA/DNA separation or stored at −80 °C.
5
Single-cell Multiomics of Early Embryo
6
Methylation Profile Generation Protocol
7
Methylation of Isolated Nuclei Protocol
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Cell pellet was resuspended with cold lysis buffer: 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) –NaOH pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1× proteinase inhibitor (Sigma 11873580001), 0.1% Tween-20, 0.1 mg/ml bovine serum albumin (BSA), 0.1 mM EDTA (Ethylenediaminetetraacetic acid), 0.5% CA-630, incubate on ice for 5 min. Centrifugate lysis mixture at 500 × g for 5 min at 4°C to collect the nuclei. Washed the nuclei once with 1× GC buffer (NEB M0227L) then resuspend 2 million nuclei in 500 μl methylation reaction mixture: 1× GC buffer, 200 U M.CvipI (NEB M0227L), 96 μM S-adenosylmethionine, 300 mM sucrose, 0.1 mg BSA, 1× proteinase inhibitor, 0.1% Tween-20. Incubate the reaction for 3 hr at 37, add 96 μM SAM (S-Adenosylmethionine) and 20 U M.CvipI per hour. Centrifugate at 500 × g for 10 min at 4°C to collect nuclei, wash the nuclei once with chilled HEPES–NaOH pH 7.5 and centrifugate to collect nuclei.
8
Genomic DNA Analysis using MAPit-BGS
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9
Nanopore-based NOMe-seq for Chromatin Profiling
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NOMe-seq was performed on the cells with adjustments for nanopore sequencing. Cells were collected by trypsinization, then nuclei were extracted by incubating in resuspension buffer (100 mM Tris-Cl, pH 7.4, 100 mM NaCl, 30 mM MgCl2) with 0.25 % NP-40 for 5 minutes on ice. Intact nuclei were collected by centrifugation for 5 minutes at 500xg at 4°C. Nuclei were subjected to a methylation labeling reaction using a solution of 1x M. CviPI Reaction Buffer (NEB), 300 mM sucrose, 96 µM S-adenosylmethionine (SAM; New England Biolabs, NEB), and 200 U M. CviPI (NEB) in 500 µL volume per 500,000 nuclei. The reaction mixture was incubated at 37 °C with shaking on a thermomixer at 1,000 rpm for 15 minutes. SAM was replenished at 96 µM at 7.5 minutes into the reaction. The reaction was stopped by the addition of an equal volume of stop solution (20 mM Tris-Cl, pH 7.9, 600 mM NaCl, 1% SDS, 10 mM disodium EDTA). Samples were treated with proteinase K (NEB) at 55 °C for > 2 hours, and DNA was extracted via phenol:chloroform extraction and ethanol precipitation. After proteinase K treatment, and in all following steps, samples were handled with care using large orifice pipette tips to avoid excessive fragmentation of DNA.
10
Nanopore-based NOMe-seq for Chromatin Profiling
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