Luciferase expression vector pGL4.14[luc2/Hygro] with or without a −146/+46 promoter fragment of the AS3MT gene (~6 μg) was incubated with 20 units of M.SssI methyltransferase (New England Biolabs, Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (New England Biolabs) for 6 h. As a negative control, the reporter construct was prepared in the absence of the M.SssI methyltransferase. The M. SssI methyltransferase was heat inactivated at 65 °C for 20 min according to the manufacturer’s instructions. The constructs were ethanol precipitated before transfection into cells and determination of luciferase activity. The methylation-sensitive restriction enzyme HpaII (New England Biolabs) was used to analyze the methylation status of each construct (data not shown).
M sssi methyltransferase
Manufactured by New England Biolabs
Sourced in United States
M.SssI methyltransferase is an enzyme that catalyzes the methylation of cytosine residues in DNA. It recognizes and methylates the dinucleotide sequence 5'-CG-3'.
Automatically generated - may contain errors
Lab products found in correlation
Ez dna methylation kit, by Zymo Research (3 mentions)
Hpaii, by New England Biolabs (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Gibson assembly master mix protocol, by New England Biolabs (2 mentions)
Proteinase k, by Roche (1 mentions)
Whole genome amplification, by Qiagen (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Tianamp micro dna kit, by Tiangen Biotech (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Pcpgfree promoter lucia vector, by InvivoGen (1 mentions)
Hpych4iv digestion, by New England Biolabs (1 mentions)
Hpaii, by Thermo Fisher Scientific (1 mentions)
S adenosylmethionine, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Genomiphi hy kit, by GE Healthcare (1 mentions)
17 protocols using m sssi methyltransferase
1
Methylation Analysis of Luciferase Reporter
2
DNA Methylation Analysis Protocol
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One microgram of genomic DNA per sample was modified using the EZ DNA methylation kit (Zymo Research Corp, Irvine, US) according to the manufacturer’s instructions. Leukocyte DNA from healthy women was used as a negative control for methylation, while in vitro methylated leukocyte DNA produced using M. SssI methyltransferase (New England Biolabs, Ipswitch, USA) was used as a positive control.
3
Nuclei Isolation and M.SssI Methylation
4
Genome-wide DNA Methylation and Expression Analysis
5
Targeted DNA Methylation Analysis of Embryos
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We selected 20 target from the methylKit analysis to validate the WGBS data and analysed these targets in F2 and F3 generation embryos (Supplemental Figure S1 ). One target (atm) did not show consistent results following standard curve analysis and was discarded. We used 5 replicate pools of embryos per exposure group per generation with this analysis. We used the BisPCR2 method59 (link), which was adapted at our lab and is extensively described16 (link), and details can be found in SI materials and methods. Primers were developed using the online Bisearch tool (http://bisearch.enzim.hu/ ), and validated for specificity and amplicon size by gel electrophoresis, as described previously (Supplemental Table S1 )16 (link). Each primer was validated with standard curve analysis using different ratios of unmethylated DNA to fully methylated DNA. Unmethylated DNA was produced by means of whole genome amplification (Qiagen, Germany) and methylated DNA by M.SssI methyltransferase (New England Biolabs, US)16 (link). Downstream bioinformatics analysis was performed similarly as with WGBS. Statistical analysis was performed with Seqmonk (v1.36), using the logistic regression filter, with Benjamini-Hochberg FDR correction, with a methylation cut-off of 10%.
6
TagMan-based QMSP for DNA Methylation Analysis
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TagMan-based QMSP (MethyLight) [36 (link)] method was used to determine the methylation level of HCCs. We used type II collagen gene (COL2A) for an internal reference gene by amplifying the non-CpG sequences. Each sample was analyzed three times. The genomic DNA treatment with M.Sss I methyltransferase (New England Biolabs, Beverly, MA) was used as positive control. The QMSP reactions were done as our previous report [37 (link)]. The relative DNA methylation was determined based on the threshold cycles (Ct) of the gene of interest and of the internal reference gene (COL2A). The relative DNA methylation level [sample_gene/sample_COL2A] was calculated by the ΔCt method [36 (link), 38 (link)]. Testing results with Ct-value of COL2A greater than 40 were determined as detection failure.
7
Genomic DNA Extraction and Methylation Analysis
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Genomic DNA from frozen tissues was extracted using a TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. DNA from cervical liquid-based cell specimens was isolated by phenol/chloroform extraction. One microgram of genomic DNA per sample was modified using the EZ DNA methylation kit (Zymo Research Corp, Irvine, US) according to the manufacturer's instructions. Leukocyte DNA from healthy women was used as a negative control for methylation, while in vitro methylated leukocyte DNA produced using M. SssI methyltransferase (New England Biolabs, Ipswitch, USA) was used as a positive control.
8
DNA Extraction and Bisulfite Modification
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Processing of cervical scrapings and assessment of the DNA's structural integrity were as described previously.19 Genomic DNA was extracted from the cervical exfoliated cells using the TIANamp Micro DNA Kit (Tiangen Biotech Co. Ltd, Beijing, China) following the manufacturer's recommendations. DNA concentrations and 260/280 ratios were measured using a Nanodrop ND‐1000 spectrophotometer (Thermo Fisher Scientific Inc, Valencia, CA). A 260/280 ratio around 1.8 to 2.0 and the capability to produce amplicons of at least 300 base pairs (bp) using a BIOMED2 multiplex PCR21 was required for all DNA samples. Sodium bisulfite modification of denatured genomic DNA was performed as previously reported.22 One microgram DNA was treated with sodium bisulfite using EZ DNA Methylation‐Gold kit according to manufacturer's instructions of (Zymo Research, Irvine, CA) and eluted in 100 μL to obtain 10 ng/μL. Samples were randomly distributed among DNA isolation batches and were again randomized across multiple bisulfite treatments. Leukocyte DNA from five healthy women was pooled and used as negative control for methylation, whereas in vitro methylated leukocyte DNA (IV), produced using M. SssI methyltransferase (New England Biolabs, Ipswitch, MA), served as a positive control.
9
Zfp423 Promoter Mutagenesis and Methylation
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Zfp423 promoter (−1037/−1002) was amplified by PCR and cloned into the firefly luciferase reporter pCpGfree-promoter-Lucia vector (InvivoGen). A one-step PCR-based mutagenesis technique was used to generate site-specific mutation [28 (link)] and produce a mutated construct. One complementary pair of primers was designed that contained the desired mutation, replacing the cytosine at position −1016 with adenine. The wild-type and mutated constructs were transformed into E. coli GT115 cells. These cells were purchased from InvivoGen and are mycoplasma-free. In vitro methylation was performed using M.SssI methyltransferase following the manufacturer’s protocol (New England BioLabs). Unmethylated wild-type and mutated constructs were obtained in the absence of M.SssI. Methylation was confirmed by resistance to HpyCH4IV digestion (New England Biolabs). After 48 h, firefly and Renilla luciferase activity were assayed using a luciferase reporter assay kit, as reported in the previous paragraph.
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Zfp423 promoter (−1037/−1002) was amplified by PCR and cloned into the firefly luciferase reporter pCpGfree-promoter-Lucia vector (InvivoGen). A one-step PCR-based mutagenesis technique was used to generate site-specific mutation [28 (link)] and produce a mutated construct. One complementary pair of primers was designed that contained the desired mutation, replacing the cytosine at position −1016 with adenine. The wild-type and mutated constructs were transformed into E. coli GT115 cells. These cells were purchased from InvivoGen and are mycoplasma-free. In vitro methylation was performed using M.SssI methyltransferase following the manufacturer’s protocol (New England BioLabs). Unmethylated wild-type and mutated constructs were obtained in the absence of M.SssI. Methylation was confirmed by resistance to HpyCH4IV digestion (New England Biolabs). After 48 h, firefly and Renilla luciferase activity were assayed using a luciferase reporter assay kit, as reported in the previous paragraph.
10
In vitro DNA methylation and oxidation
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In vitro methylation of pOct4-GFP plasmid DNA was performed using M.SssI methyltransferase (New England Biolabs) according to the manufacturer's instructions. The methylation status of the plasmid was tested by HpaII and MspI (Fermentas) digestion.
For the in vitro oxidation, GFP-TET1CD or GFP-TET1CDmut (H1652Y, D1654A) was purified from mammalian cells. In detail, HEK293T cells were transfected with an expression construct for GFP-TET1CD/TET1CDmut and immunoprecipitation was carried out using GBP-Ni-NTA beads. Proteins were eluted using imidazole. The in vitro methylated plasmid was diluted in TET reaction buffer (50 mM HEPES pH 8.0, 75 μM Fe(II), 2 mM Sodium-Ascorbate, 1 mM Di-Sodium-Ketoglutarate (39 (link))) and added to the purified GFP-TET1CD.
For the in vitro oxidation, GFP-TET1CD or GFP-TET1CDmut (H1652Y, D1654A) was purified from mammalian cells. In detail, HEK293T cells were transfected with an expression construct for GFP-TET1CD/TET1CDmut and immunoprecipitation was carried out using GBP-Ni-NTA beads. Proteins were eluted using imidazole. The in vitro methylated plasmid was diluted in TET reaction buffer (50 mM HEPES pH 8.0, 75 μM Fe(II), 2 mM Sodium-Ascorbate, 1 mM Di-Sodium-Ketoglutarate (39 (link))) and added to the purified GFP-TET1CD.
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