Supplementary Figure S1 Supplementary Figure S2 Supplementary Figure S3. Supplementary Figure S4

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1 Supplementary Figure S1 Supplementary Figure S2 Supplementary Figure S3 Supplementary Figure S4 Supplementary Figure S5 Supplementary Figure S6 Supplementary Figure S7 Supplementary Figure S8 Supplementary Table S1 Supplementary Table S2 Supplementary Table S3 Supplementary Table S4 Supplementary Methods Recombinant mtet1 ( ) purification and activity test Recombinant mtet1 substrate selectivity assay Comparison of 5mC conversion and labeling efficiency by using the one-pot mtet1/β-gt and sequential mtet1/β-gt method Quantitative analysis of 5mC oxidation using LC-MS/MS Validation of stability of the glucosylated 5hmC in DNA Genomic snapshot of MeDIP-Seq, TAmC-Seq and non-enriched Input genomic DNA read densities across the Pcdha locus Genomic snapshot of MeDIP-Seq, TAmC-Seq and non-enriched Input genomic DNA read densities across the Anmy1/Dusp28/Rnpepl1 locus Global correlation of affinity approaches and bisulfite sequencing Quantitative analysis of standards as working calibrations using LC-MS/MS Quantitative analysis of 5mC oxidation using LC-MS/MS Summary of average percent methyation 1kb bins with read depth 5 in TAmC-Seq and MeDIP-Seq Summary of sequence reads analyzed The Huisgen cycloaddition (click) reaction and pull down S1

2 Supplementary Figure S1. Recombinant mtet1 protein ( ) purification and activity test. (a) Schematic diagrams of the mouse Tet1 protein. The major domains are shown as colored cube as indicated. (b) Coomassie-stained SDS-PAGE gel containing recombinant Flag-mTet1 purified from SF9 insect cells. (c) Recombinant mtet1 in vitro activity test using MALDI-TOF. The model dsdna sequence is shown. The MALDI-TOF characterization on 12mer DNA is shown with the calculated and observed molecular weight indicated. S2

3 Supplementary Figure S2. Recombinant mtet1 substrate selectivity assay. (a) The sequence of 32mer-44mer dsdna is shown. The cytosine in red indicates the modification site (C, 5mC or 5hmC). (b) Schematic diagram of the selectivity assay. (c) The assay products were separated using 16% urea denatured acrylamide gel. The gel was scanned at 563nm and fluorescence at 582nm (the labeled dye on 5hmC) was monitored, and then stained with SYBER Green. The results indicate mtet1 could recognize and oxidize 5mC to 5hmC on different combinations of 5mC and 5hmC at CpG site, such as hemi-5mc, full-5mc, or hemi-5mc/hemi-5hmc modification, ensuring that 5mC in various contexts in genomic DNA could be efficiently recognized and oxidized by mtet1 for further labeling. S3

4 Supplementary Figure S3. Comparison of 5mC conversion and labeling efficiency between the one-pot mtet1/β-gt protocol and the sequential mtet1/β-gt method. 32mer dsdna containing one full 5hmCpG site in the middle which has been labeled with N3-Glc and biotin was employed to generate the standard curve, and the concentration of each spot is as indicated. 32mer dsdna containing one single 5mC (exactly the same DNA sequence with the standard dsdna except that the full 5hmC site was replaced by a single 5mC site) was used as mtet1 substrate, and various concentrations of recombinant mtet1 were used as indicated. The products were diluted in gradient for dot blot. The conversion ratio was evaluated by dot blot assay of the attached biotin. S4

5 Supplementary Figure S4. Quantitative analysis of 5mC oxidation using LC-MS/MS. The nucleosides were quantified using the nucleoside to base ion mass transitions. Most 5mC are converted to N 3-5gmC with minimum 5fC and 5caC (less than 2%) observed and ~3% 5mC remaining in the one-pot protocol (see Supplementary Table 1) S5

6 Supplementary Figure S5. Validation of stability of the glucosylated 5hmC in DNA. Mass spectrometry characterization of the model reaction with a 5hmC-containing 9mer DNA annealed to a 11mer DNA. The reactions were monitored by MALDI-TOF with the calculated molecular weight and observed molecular weight indicated. The additional peak (m/z=2804.4) labeled in the center and bottom panels is not the unreacted 5hmC-containing strand. The -150 peaks exist for all other oligonucleotides ( and ). We think these are likely fragments from mass spec experiments or side products derived from DNA synthesis. S6

7 Supplementary Figure S6. Genomic snapshot of MeDIP-Seq, TAmC-Seq and non-enriched Input genomic DNA read densities across the Pcdha locus (chr18: 37,056,628-37,322,944). S7

8 Supplementary Figure S7. Genomic snapshot of MeDIP-Seq, TAmC-Seq and non-enriched Input genomic DNA read densities across the Ankmy1/Dusp28/Rnpepl1 locus (chr1: 94,755,795-94,826,014). S8

9 Supplementary Figure S8. Global correlation of affinity approaches and bisulfite sequencing. Genome-wide correlation of TAmC-seq (top, middle) and MeDIP-seq (bottom) with bisulfite sequencing. S9

10 Supplementary Table S1. Quantitative analysis of standards as working calibrations using LC-MS/MS. The standard curves of da, 5mdC, 5hmdC, 5fdC and 5cadC are obtained from various concentrations of pure nucleoside standards. Samples da 5mdC 5hmdC 5fdC 5cadC Conc. (µm) Counts Conc. (µm) Counts Conc. (µm) Counts Conc. (µm) Counts Conc. (µm) Counts Standard E E E E Standard E E E E Standard E E E E Standard E E E E Standard E E E E S10

11 Supplementary Table S2. Quantitative analysis of 5mC oxidation using LC-MS/MS. Quantification was performed by comparison with a standard curve obtained from pure nucleoside standards running at the same batch of samples. The ratio of 5mdC, 5hmdC, 5fdC and 5cadC were calculated based on the calculated concentrations. 5hmC labeled with glucose (or 6- N 3 -glucose) was not monitored since they elute outside of our HPLC separation range. However, we can directly monitor changes of 5mdC, 5hmdC, 5fdC, and 5cadC to determine the oxidation and protection efficiency. Quantitative analysis data of samples that were treated with different protocols as indicated. Samples da 5mdC 5hmdC Counts Conc. (µm) Counts Conc. (µm) 5mdC/dA (%) Counts Conc. (µm) 5hmdC/dA (%) mesc DNA E E E-02 β-gt+udp-glc E N/A N/A N/A One-pot mtet1/β-gt E E E-03 Samples 5fdC 5cadC 5mdC Counts Conc. (µm) 5fdC/dA (%) Counts Conc. (µm) 5cadC/dA (%) Oxidation ratio (%) mesc DNA N/A N/A N/A N/A N/A N/A N/A β-gt+udp-glc N/A N/A N/A N/A N/A N/A N/A One-pot mtet1/β-gt E E E E a a : The ratio indicates the most 5mC is oxidized and labelled with biotin. S11

12 Supplementary Table S3. Summary of average percent methyation 1kb bins with read depth 5 in TAmC-Seq and MeDIP-Seq. Average % methylation TAmC-Seq, all MeDIP-Seq, all TAmC-Seq+MeDIP-Seq overlap TAmC-Seq specific MeDIP-Seq specific All CpGs S12

13 Supplementary Table S4. Summary of sequence reads analyzed. Combined unique, non-duplicate reads J1 mes TAmC-Seq J1 mes MeDIP J1 mes Input Replicates unique, non-duplicate reads J1 mes TAmC-Seq rep J1 mes TAmC-Seq rep J1 mes TAmC-Seq rep J1 mes MeDIP (ERR031627_1) J1 mes MeDIP (ERR031630_1) J1 mes Input S13

14 Supplementary Methods The Huisgen cycloaddition (click) reaction and pull down The Huisgen cycloaddition (click) reaction and pull down was processed according to the previous protocol 26. The click chemistry was performed in 40 µl solution containing N 3 -Glc labeled DNA fragments and 150 µm dibenzocyclooctyne with disulfide biotin linker. The reaction mixture was incubated for 2 h at 37 C, and then purified by Qiagen nucleotide removal kit. The DNA fragments were eluted in 50 µl H 2 O, and applied immediately to the Invitrogen Dynabeads MyOne Streptavidin C1 beads for pull down assay following the general immobilization of nucleic acids from the manufacturer s manual. Subsequently, the beads were incubated in 50 µl of 100 mm DTT in H 2 O for 2 h at room temperature to cleave the disulfide bonds. The supernatant was then applied to Bio-Rad Micro Bio-Spin 6 spin columns to remove DTT. The DNA fragments were finally purified with the Qiagen MinElute PCR Purification kit and eluted into EB buffer, which were ready for DNA sequencing library build up. S14