Cells were trypsinized and centrifuged for 7 min at 160×g, then washed in ice-cold PBS and resuspended in 1 mL ice-cold Nuclei Buffer (10 mM Tris,pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA and 1% IGEPAL, plus protease inhibitors) per 5 × 106 cells and incubated on ice for 10 min. To obtain the nuclei, cells were dounce-homogenized for 15 times and incubated on ice for about 45 min, until single nuclei were visualized under the microscope. Nuclei were recovered by centrifugation at 500×g for 5 min and washed in Nuclei Wash Buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1 mM EDTA containing protease inhibitors). Freshly prepared nuclei (2 × 105 cells) were resuspended in 1× M.CviPI reaction buffer (NEB), then treated for 15 min with 150 U of M.CviPI (NEB M0227B; 50,000 U mL−1) in 15 µL 10× reaction buffer, 45 µL 1 M sucrose and 0.75 µL SAM in a volume of 150 μL. Reactions were quenched by the addition of an equal volume of Stop Solution (20 mM Tris–HCl [pH 7.9], 600 mM NaCl, 1% SDS, 10 mM EDTA, 400 µg mL−1 Proteinase K) and incubated at 55 °C overnight. DNA was purified by phenol/chloroform extraction and ethanol precipitation. The resulting DNA was used to perform either whole genome bisulfite sequencing or amplicon sequencing.
M cvipi reaction buffer
Manufactured by New England Biolabs
M.CviPI Reaction buffer is a specialized buffer solution designed for use with the M.CviPI DNA methyltransferase enzyme. It provides the optimal ionic conditions and pH for the enzyme to effectively modify DNA sequences. The buffer composition ensures efficient and reliable performance of the M.CviPI enzyme during DNA methylation reactions.
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Lab products found in correlation
5 protocols using m cvipi reaction buffer
1
Nuclei Isolation and DNA Methylation Mapping
2
Single-cell Multiomics of Early Embryo
3
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.
4
Nanopore-based NOMe-seq for Chromatin Profiling
5
Single-cell Multiomics of Early Embryo
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Single-cells were flow-sorted (E6.5 and E7.5 stages, using a BD Influx or BD Aria III) or manually picked when cell numbers were too low (E4.5, E5.5). Cells were isolated into 96 well PCR plates containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl−1 RNasein (Promega), 0.1% IGEPAL CA-630 (Sigma)). Samples were incubated for 15 minutes at 37°C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at -80°C prior to processing. Poly-A RNA was captured on oligo-dT conjugated to magnetic beads and amplified cDNA was prepared according to the G&T-seq32 (link) and Smartseq2 protocols33 (link). The lysate containing gDNA was purified on AMPureXP beads before bisulfite-seq libraries were prepared according to the scBS-seq protocol34 (link).
A subset of embryo cells were processed for scRNA-seq only (1,419 cells after QC). These followed the same protocol but we discarded the gDNA after separation.
A full step-by-step protocol for scNMT-seq is available online:dx.doi.org/10.17504/protocols.io.6jnhcme .
A subset of embryo cells were processed for scRNA-seq only (1,419 cells after QC). These followed the same protocol but we discarded the gDNA after separation.
A full step-by-step protocol for scNMT-seq is available online:
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