Predominance of N6-Methyladenine-Specific DNA Fragments

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Predominance of N6-methyladenine-specific DNA fragments enriched by multiple immunoprecipitation Xiaoling Liu, Weiyi Lai, Ning Zhang, and Hailin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01087 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 15, 2018

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Analytical Chemistry

Predominance of N6-methyladenine-specific DNA fragments enriched by multiple immunoprecipitation Xiaoling Liu†,‡, Weiyi Lai†,‡, Ning Zhang†,‡, and Hailin Wang†,‡,§,* †

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 10085, China ‡ University of Chinese Academy of Sciences, Beijing, 10085, China § Institute of Environment and Health, Jianghan University, Wuhan, 430056, China

ABSTRACT: N6-methyladenine (6mA) is a re-discovered DNA modification in eukaryotic genomes. To explore the distribution and functions of 6mA, it is of paramount option to use immunoprecipitation to select 6mA-containing DNA fragments for genomewide sequencing. Presumably, most of 6mA-free fragments is removed, and the co-pulling down of the residual is stochastic and sequence-independent and thus they should not be called as peaks by computation. Surprisingly, here we show the predominance of 6mA-free fragments in the pulled-down fractions. By taking advantage of the sub-micromolar affinity of the antibodies, we further develop an elegant, multiple-round immunoprecipitation (MrIP) approach, and show that 6mA-containing fragments can be enriched over 9100 folds and dominate in the final pulled-down fractions. This biochemical approach would greatly reduce the peak calling bias, which is caused by handling of dominated 6mA-free DNA fragments with an assumption-based algorithm computation, and facilitates 6mA-pertinent datamining. The MrIP concept is extendable for the genome-wide sequencing of diverse DNA modifications.

DNA N6-methyladenine (6mA) is one of dominant epigenetic modifications in prokaryotes,1-3 and is a potential epigenetic mark in eukaryotes.4-6 In some bacteria, DNA N6-adenine methyltransferase is critical for their viability.7-9 Recently, we and other groups showed the prevalence of 6mA in the genomes of higher eukaryotes, such as Chlamydomonas algae,10 Caenorhabditis elegans,11 Drosophila melanogaster.12 Later, DNA 6mA was also found in zebrafish13 and Xenopus Laevis,14 and probably in mouse embryonic stem cells15,16 and pig embryos.13 Immunoprecipitation (IP) is an invaluable and common tool required for both locus-specific and genome-wide profiling of DNA methylations.10,12,14,17,18 Ideally, anti-6mA antibody only recognizes genomic DNA 6mA. If so, 6mA-free DNA fragments should be completely removed through anti6mA antibody-based immunoprecipitation (6mA-IP). In practice, avidity, affinity, and specificity of the antibodies, protein A/G, and microbeads involved in the IP could cause nonspecific binding of 6mA-free DNA fragments. It is known that epigenetic DNA modifications are often written and enriched in specific sequences or loci of genomes.19-21 Presumably, the stochastic introduction of 6mA-free fragment by non-specific binding is independent of DNA sequences during the immunoprecipitation. Therefore, it believes that the peaks are selectively called only for 6mA-specific sequences by computation algorithm.22 However, the accuracy of peak calling is highly dependent on the computation algorithm and individual skills,23,24 and it remains elusive how to verify the computation-inferred judgements experimentally.25 In this work, we exploited an advanced analytical technology, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)12,26,27 to evaluate 6mA-

IP. Benefiting from excellent sensitivity (low limit of detection of mass: attomole level) and structure-dependent measurement of the developed UHPLC-MS/MS approach, we could detect 6mA accurately in the final pulled-down DNA (down to one nanogram). Therefore, it is feasible to evaluate 6mA-IP using UHPLC-MS/MS throughout whole immunoprecipitation procedure. Moreover, we demonstrate that a large portion of the pulled-down DNA does not contain any 6mA, particularly for dealing with the genomes with 6mA of very low abundance. To eliminate possible bias caused by the predominance of 6mA-free fragments, we for the first time designed an elegant, multiple-round 6mA-IP to allow the possible predominance of 6mA-specific fragments in the final pulled-down fractions. EXPERIMENTAL SECTION Chemicals and materials. Lambda DNA (D152A) was purchased from Promega Systems Ltd (Wisconsin, USA). The DNA (D152A) was isolated from the infected GM119 strain of E. coli, lacking the major DNA N6-adenine methyltransferase gene (dam28). Five anti-6mA rabbit IgG antibodes were respectively purchased from Synaptic Systems (202003, Goettingen, Germany), Abcom company (two from this company, monoclonal, 190886; polyclonal, 151230; Cambridge, UK), Millipore company (ABE572, Darmstadt, Germany) and Active Motif company (61495, Carlsbad, North America). Protein A/G Agarose beads (BD0048) were purchased from Bioworld Technology CO., Ltd (Nanjing, China). Glycogen (R0561) was ordered from ThermoFisher Scientific (MA, USA). Deoxyribonuclease I (DNase I) and calf intestinal alkaline phosphatase (CIP) were obtained from New England Biolabs (Ipswich, MA). Snake venom phosphodiesterase I (SVP)

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was purchased from Washington Biochemical Corporation (Lakewood, USA). Ultrapure water was prepared in our lab using ELGA PureLab-Ultra purifier (High Wycombe, UK). 2′Deoxyguanosine monohydrate (dG·H2O, ≥ 99%), 2′deoxycytidine (dC), LC-MS grade methanol (Fluka) and formic acid (for mass spectrometry, ~98%) were ordered from Sigma-Aldrich (St. Louis, MO, USA). N 6methyldeoxyadenosine (6mA) were ordered from Santa Cruz Biotechnology, Inc. (Texas, USA). DNA fragmentation. DNA was fragmented by ultrasonication using the Covaris S2 Focused-ultrasonicator (Covaris, Inc., Woburn, Massachusetts, USA). First, DNA (1.05.0 µg) was diluted in 130 µl TE buffer (1.0 Tris-HCl, pH7.5, plus 0.1 mM EDTA (ethylenediaminetetraacetic acid)), and then transferred into a Covaris microTUBE. The DNA was sheared to 150 bp using the parameters: Bath temperature: 5°C; 5 cycles (Duty cycle: 10%; Intensity: 5; Cycles per burst: 100; Time: 60s). The sheared DNA was stored up to 2 months at -20ºC. DNA 6mA-immunoprecipitation. We performed DNA 6mA-immunoprecipitation (6mA-IP) using rabbit anti-6mA IgG antibodies. First, the fragmented DNA (0.5-10 µg) was diluted in 1.0 × IP buffer of 300 µL (10 mM phosphate buffer, pH 7.2, plus 140 mM NaCl, 0.05% Triton X-100) and denatured at 95 °C for 10 min. Then, the denatured DNA was immediately placed on ice and subsequently mixed with anti6mA antibody (0.25-5.0 µg, 0.5 µg/µL) for 4.0 h incubation at 4 °C with overhead shaking. A portion of fragmented DNA was taken as Input fraction, which did not undergo any step involved in the immunoprecipitation, for further UHPLCMS/MS characterization and analysis. Simultaneously, the protein A/G Agarose beads (10-50 µL, every 10 µL protein A/G Agarose beads can bind 1.0-2.0 µg antibody) were prewashed twice with 500 µL 1.0 × IP buffer, and re-suspended in 20 µL 1.0 × IP buffer. After the incubation of the anti-6mA antibody with DNA, the protein A/G Agarose beads were added and incubated for 2.0 h at 4 °C with gentle rotation. The supernatant was discarded, and the beads were then washed three times using 500 µL 1.0 × IP buffer. During each washing, the beads were shaken for 10 min at 4 °C. After washing, the beads were mixed with an elution buffer (200 µL, 5.0 mM Tris-HCl, pH 8.0, 0.5% SDS, and 0.2 mg/mLproteinase K). To release the antibody-bound DNA, the mixed solution was treated with the proteinase K (included in the elution buffer) at 55°C. To prevent sedimentation of the beads, the solution was put in a shaking heat block and shaken at 800 rpm. After 3.0 h digestion, the beads were removed by centrifugation at the speed of 1000 rpm for 1.0 min, and the eluted solution containing the released DNA was treated by phenol/chloroform/isoamyl alcohol (v/v/v, 25:24:1), and the organic phase was discard. The aqueous solution retaining the released DNA was added with 1.0 µL glycogen (20 mg/mL) and 0.1 volume of 3.0 M sodium acetate (pH 5.2), and mixed with ethanol of three volume. Then, the solution was stored at -20 °C for 2.0 h to facilitate DNA precipitation. Finally, the DNA pellet was centrifuged at 12,000 rpm for 20 min at 4 °C, and washed with ethanol/water (7:3, v/v) three times. The recovered DNA pellet was dried and re-dissolved in 30 µL of ddH2O. For the multiple-round IP (MrIP), the enriched DNA was suffered from next round pulled-down until the 6mA-specific

DNA fragments were dominant in the pulled-down DNA fragments as evaluated by UHPLC-MS/MS analysis.

UHPLC-MS/MS analysis. The DNA was enzymatically digested into 2’-deoxynucleosides by a mixture of DNase I, calf intestinal phosphatase, and snake venom phosphodiesterase I at 37 °C for 6.0 h.26,27 After ultrafiltration, the digested DNA was subjected to UHPLC-MS/MS analysis. The UHPLC-MS/MS analysis was performed on an Agilent 1290 II UHPLC system coupled with an ESI-triple quadrupole mass spectrometer (6470, Agilent Technologies, Santa Clara, CA). A ZORBAX SB-Aq column (2.1 × 100 mm, 1.8 µm particle size, Agilent, USA) was employed for the separation of monodeoxynucleosides. The mass spectrometer was operated under positive ionization using multiple reactions monitoring (MRM) mode. The selective MRM transitions were monitored as follows: m/z 266→150 for 6mA, and m/z 268→152 for dG. The fragmentation voltage for all the MRM transitions were set at 90 V to allow efficient transit of precursor ions. Nitrogen gas was used for nebulization and desolvation. The nebulization gas pressure was set at 40 psi, and the temperature and the flow rate of desolvation gas were set at 300 °C and 9.0 L/min, respectively. High purity nitrogen (99.999%) was used as collision gas. The other conditions were adopted as previously.12,26,27 RESULTS AND DISCUSSION We firstly evaluated five anti-6mA IgG antibodies (listed in Table S1). To test IP efficiency of each antibody, we used one lambda DNA, which was extracted from an infected GM119, an E. coli strain lacking dam. Since dam protein is the dominant DNA N6-adenine methyltransferases in E coli,28 the lambda DNA extracted from dam-depleted GM119 should have DNA 6mA of low abundance, named as L-6mA lambda DNA. As detected by UHPLC-MS/MS, the frequency of 6mA for L-6mA lambda DNA is estimated to be 0.68 6mA modification per 104 deoxynucleotides (nts). Prior to IP, the L-6mA lambda DNA was fragmented into ~ 150 nt by ultra-sonication and denatured thermally as single-strand (ss) DNA (Figure 1A). By the use of fragmented and denatured L-6mA lambda DNA and anti-6mA rabbit IgG antibodies, we performed 6mA-IP under optimized conditions. As detected by UHPLCMS/MS, we observed that all the five tested antibodies displayed a capacity of enriching 6mA fragments from the L6mA lambda DNA (Figure 1B and 1C). The pulled-down DNA fragments display an elevate level of 6mA (0.036-0.25 6mA per 100 nts, Figure 1). Interestingly, the anti-6mA antibodies of the AM and Mp (abbrev. ref to Table S1) were highly specific, displaying the highest enrichment (26-37 folds). In contrast, both the antibodies of SY and Abc-poly only displayed a moderate enrichment (12-18 folds), but the mass of the pulled-down DNA (28.9-30 ng) was more than 12 times higher than that obtained using the antibodies of AM and Mp (~2.1-2.4 ng DNA, Figure S1). To simplify the following mathematic estimation, we assumed that 6mA was randomly distributed among the pulleddown fragments, and each ssDNA fragment should contain either one or none 6mA modification. Considering the average length of fragmented DNA (150 nts, Figure 1A), it estimated that the frequency of 6mA was about 0.67 modification per 100 nts if every fragment contained one 6mA. Essentially, the pulled-down DNA fraction was enriched with a level of 0.25 6mA per 100 nts maximally (Figure 1C), and thus the pulleddown DNA fragments of 62.7% at least did not contain any 6mA modification (Figure 1D). If some fragments contained


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Analytical Chemistry more than one 6mA, the proportion of 6mA-free DNA fragments should increase. By same estimation, the percentage of 6mA-free fragments obtained from other antibodies-based IP accounted for 73.5-94.6% (Figure 1D). By the use of SY antibody of lower mass (0.25 µg), the frequency of 6mA in the pulled-down DNA increased to 0.18 6mA per 100 nts (Figure S2). The estimated percentage of 6mA-free fragments was about 73.1%.

Figure 1. The evaluation of five rabbit anti-6mA IgG antibodies for immunoprecipitation of DNA 6mA of low abundance. (A) Agarose (0.8%) gel electrophoresis analysis of fragmented lambda DNA by ultrasonication. (B, C) UHPLC-MS/MS chromatograms (B) and quantification (C) of 6mA. (D) Estimated percentage of 6mA-free fragments in the pulled-down fractions. The chromatograms of 6mA were normalized against dG. L-6mA lambda DNA was used for IP test. Antibody, 3.0 µg; DNA input, 5.0 µg. The abbrev. names of the anti-6mA antibodies were listed in Table S1.

Regarding the observed predominance of 6mA-free fragments in the final pulled-down DNA fractions, it was plausible to speculate that these 6mA-free fragments co-pulled down with 6mA-specific fragments could be falsely presented as the 6mA peaks in the datamining of the genome-wide sequencing, and true 6mA peaks would be easily masked due to their low abundance in the final pulled-down fractions. The next question was how to reduce the 6mA-free fragments in the final pulled-down DNA fractions. To this regard, there were two routine ways to try: 1) crossing-link chemistry; 2) biochemical synthesis of new antibodies. However, chemical-induced29 or photo-activated crossing-link30-32 would introduce more non-specific binding if the reactions were not well controlled, and the synthesis of new antibodies with requested affinity and selectivity might take years with a gambling on success. Here we thought about the third way, which requires neither the crossing-link nor synthesis of new antibodies. Based on preliminary immunoprecipitation results (Figure 1), the tested anti-6mA antibodies could enrich 6mA from L-lambda DNA with a factor of 5.3-37 folds (Figure 1), whereas 6mA-free fragments remained to predominate in the final pulled-down fractions. We inferred that these antibodies might have sub-micromolar affinity against 6mA in the presence of abundant 6mA-free fragments. Considering the moderate affinity and low selectivity, the IP looked like a liquid phase distribution-based extraction.33 If so, multiple-round IP

should be very helpful to get 6mA-specific fragments of high proportion in the final pulled-down fractions. Therefore, we designed a multiple-round 6mA-IP procedure (6mA-MrIP, Scheme 1). Briefly, we used one anti-6mA antibody to enrich 6mA-fragments through iterative immunoprecipitation of genomic DNA until 6mA-fragments dominate in the final pulleddown DNA fractions. In this MrIP, the enrichment of 6mA was evaluated using UHPLC-MS/MS assay.

Scheme 1. Schematic illustration of multiple-round DNA 6mA-IP procedure By the use of this 6mA-MrIP procedure, we tested two antibodies (SY and AM) for enriching 6mA-specific fragments from fragmented and denatured L-lambda DNA. Indeed, we observed a significant improvement on the enrichment of 6mA-fragments. The level of 6mA in the 2° enriched DNA was about 0.71 modification per 100 nts for SY antibody (Figure 2A) and 1.27 modifications per 100 nts for AM antibody (Figure 2B), respectively, surpassing the 6mApredominant limit (0.67 6mA per 100 nts). Overall, the final pulled-down DNA fragments contained one or two 6mA modifications per DNA fragment.

Figure 2. Two-round 6mA-MrIP of L-6mA lambda DNA by the rabbit anti-6mA antibody of SY (A) and AM (B). UHPLCMS/MS chromatograms (a) and quantification (b) of 6mA and the enriched DNA mass (c) by the two antibodies. The chromatograms of 6mA were normalized against dG. Antibody: 5.0 µg for 1°-IP, and 1.0 µg for 2°-IP; DNA input, ~ 20 µg.



We further tested the applicability of the 6mA-MrIP procedure using genomic DNA of extremely low 6mA abundance. As detected by UHPLC-MS/MS, the 6mA frequency was about 0.44 modification per 106 nts (Figure 3). Both the antibodies of SY and AM could enrich 6mA over thousands folds. Specifically, the 6mA enrichment from three-round-IP was about ~9100 folds for SY antibody (Figure 3) and ~2800 folds for AM antibody (Figure S3), respectively. Interestingly, the level of 6mA in the pulled-down DNA was about 0.40 modification per 100 nts for SY antibody. In other words, 60% of the final pulled-down DNA fractions contained one 6mA modification per fragment (~150 nts). Of note, 6mA-fragments were enriched about 34 folds by the 1° IP and further enriched about 61 folds by the 2° IP, but 6mA-free fragments remained to be dominated (Figure 3). As shown in Figure 3B, the molar quantities of 6mA-free fragments for 1° IP and 2° IP are 446 folds and 6.3 folds higher than that of 6mA-specific fragments, respectively. Only after the third round IP, the molar quantity of 6mA-specific fragments was 1.5 folds as many as that of 6mA-free fragments.

Figure 3. Three-round 6mA-MrIP of genome DNA of very low 6mA abundance. UHPLC-MS/MS chromatograms (A) and quantification (B) of 6mA and the enriched DNA mass (C) by the anti6mA antibody of SY. The chromatograms of 6mA were normalized against dG. Antibody: 5.0 µg for 1°-IP, 1.0 µg for 2°-IP, and 0.5 µg for 3°-IP; DNA input, ~ 70 µg.

The mass of the 3° enriched DNA mass was about 13.7 ng (for SY antibody), which approximately accounted for 1.67 × 1011 fragments (~150 nt). This also accounted for 2.5 × 1013 nts, which was 4700 folds as many as that of mouse genomes34 (5.3 × 109 nts). Therefore, although its mass was limited (~14 ng), the 6mA-specific DNA fraction that was finally enriched essentially provided adequate diversity for the followed construction of DNA library and genome-wide DNA sequencing. As demonstrated in this work, the advanced UHPLCMS/MS is used for quality control of anti-6mA antibodiesbased IP. It is very helpful not only for evaluation of the efficiency and specificity of the IP, but also for monitoring critical steps throughout IP. We observed that, under optimized conditions, 6mA fragments was enriched only in final pulled-down

step, but not in other operation steps (Figure S4). Noteworthy, we found that a large portion of the pulled-down DNA fragments does not contain 6mA. The finding points toward one possibility that the dominant 6mA-free DNA fragments in the pulled-down DNA will cause peaking calling bias or false representation of 6mA-fragments as computation algorithm runs. It is no doubt that the computation itself would partially eliminate some interference from these 6mA-free DNA fragments because their abundance (evaluated in individual sequences) must be low. However, it is of better choice to eliminate the 6mA-free fragments using biochemical approaches. By the use of biochemical approaches, it is possible to evaluate accurately the elimination of 6mA-free fragments (using UHPLC-MS/MS analysis). More importantly, the biochemical elimination of 6mA-free fragments greatly reduce the uncertainty caused by computation correction. To this end, we develop more-efficient, multiple-round 6mA-IP approach. Indeed, by performing the 6mA-MrIP, the 6mAfragments are enriched to be dominated, and the 6mA-free fragments can be largely eliminated from genomic DNA of extremely low 6mA abundance (Figure 3). Of note, our 6mAMrIP does not involve with any trivial and variable crosslinking. Theoretically, we can combine 6mA-MrIP with any highthroughput sequencing, including the next-generation sequencing and single-molecule real-time (SMRT) sequencing.11,15,35 Of note, SMRT sequencing has been used for direct 6mA sequencing,36-38 however, it is limited to the sequencing of unicellular 6mA at high abundance.39-41 At this moment, it is too expensive to perform genome sequencing for multicellular organisms. DnpI-assisted sequencing method was coined to locate 6mA in the sequence context of GATC, CATC and CATG.42 Essentially, a common challenge faced by these sequencing technologies and methodologies is the sequencing of a genome with 6mA of low abundance. As we demonstrated here, 6mA-MrIP provides a critical but cost-effective means for enriching 6mA up to one or more modifications per DNA fragment. By combining with this powerful 6mA-MrIP technology, it is promising to exploit these sequencing technologies to sequence the genomic 6mA of multicellular organisms at low abundance. CONCLUSION As guided by our advanced UHPLC-MS/MS analysis, we optimized 6mA-immunoprecipitation for efficiently enriching 6mA-specific fragments from the genomic DNA. Noteworthy, we found that the 6mA-free fragments are dominant in the final pulled-down DNA fragments. We further developed a multiple-round 6mA immunoprecipitation procedure. By this procedure, 6mA-specific DNA fragments are enriched approximately with one 6mA modification per fragment. Collectively, the approach is efficient, reproducible and cost-effective, and very useful to enrich genomes with a low abundance of 6mA modifications. It will greatly facilitate various 6mAspecific genome-wide sequencing.

ASSOCIATED CONTENT Supporting Information Detailed description of our experimental approach and additional experimental data (PDF) are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION


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Analytical Chemistry Corresponding Author * E-mail: [email protected]. Phone and Fax: +86-10-62849600

ACKNOWLEDGMENT This work is supported by the grants from the Ministry of Science and Technology of China (2016YFC0900300), the National Natural Science Foundation of China (21435008 and 9174321), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030200), the Key Research Program of Frontier Sciences, CAS, (QYZDJ-SSW-DQC017), and the K.C.Wong Education Foundation.

REFERENCES (1) Vanyushin, B. F.; Belozersky, A. N.; Kokurina, N. A.; Kadirova, D. X. Nature 1968, 218, 1066-1067. (2) Ratel, D.; Ravanat, J. L.; Berger, F.; Wion, D. Bioessays 2006, 28, 309-315. (3) Wion, D.; Casadesús, J. Nat. Rev. Microbiol. 2006, 4,183-192. (4) Summerer, D. Angew. Chem. Int. Ed. Engl. 2015, 54, 1071410716. (5) Luo, G. Z.; Blanco, M. A.; Greer, E. L.; He, C.; Shi, Y. Nat Rev Mol Cell Biol 2015, 16, 705-710. (6) Luo, G. Z.; He, C. Nat. Struct. Mol. Biol. 2017, 24, 503-506. (7) Stephens, C.; Reisenauer, A.; Wright, R.; Shapiro, L. Proc. Natl. Acad. Sci. 1996, 93, 1210-1214. (8) Wright, R.; Stephens, C.; Shapiro, L. J. Bacteriol. 1997, 179, 5869-5877. (9) Julio, S. M.; Heithoff, D. M.; Provenzano, D.; Klose, K. E.; Sinsheimer, R. L.; Low, D. A.; Mahan, M. J. Infect. Immun. 2001, 69, 7610-7615. (10) Fu, Y.; Luo, G. Z.; Chen, K.; Deng, X.; Yu, M.; Han, D. L.; Hao, Z. Y.; Liu, J. Z.; Lu, X. Y.; Dore, L. C.; Weng, X. C.; Ji, Q. J.; Mets, L.; He, C. Cell 2015, 161, 879-892. (11) Greer, E. L.; Blanco, M. A.; Gu, L.; Sendinc, E.; Liu, J.; Aristizabal-Corrales, D.; Hsu, C. H.; Aravind, L.; He, C.; Shi, Y. Cell 2015, 161, 868-878. (12) Zhang, G. Q.; Huang, H.; Liu, D.; Cheng, Y.; Liu, X. L.; Zhang, W. X.; Yin, R. C.; Zhang, D. P.; Zhang, P.; Liu, J. Z.; Li, C. Y.; Liu, B. D.; Luo, Y. W.; Zhu, Y. X.; Zhang, N.; He, S. M.; He, C.; Wang, H. L.; Chen, D. H. Cell 2015, 161, 893-906. (13) Liu, J. Z.; Zhu, Y. X.; Luo, G. Z.; Wang, X. X.; Yue, Y. N.; Wang, X. N; Zong, X.; Chen, K.; Yin, H.; Fu, Y.; Han, D. L.; Wang, Y. Z.; Chen, D. H.; He, C. Nat. Commun. 2016, 7, 13052-13058. (14) Koziol, M. J.; Bradshaw, C. R.; Allen, G. E.; Costa, A. S.; Frezza, C.; Gurdon, J. B. Nat. Struct. Mol. Biol. 2016, 23, 24-30. (15) Wu, T. P.; Wang, T.; Seetin, M. G.; Lai, Y.; Zhu, S.; Lin, K.; Liu, Y.; Byrum, S. D.; Mackintosh, S. G.; Zhong, M.; Tackett, A.; Wang, G. L.; Hon, L. S.; Fang, G.; Swenberg, J. A.; Xiao, A. Z. Nature 2016, 532, 329-333. (16) Schiffers, S.; Ebert, C.; Rahimoff, R.; Kosmatchev, O.; Steinbacher, J.; Bohne, A. V.; Spada, F.; Michalakis, S.; Nickelsen J.; Mgller, M.; Carell T. Angew. Chem. Int. Ed. 2017, 56, 11268-11271. (17) Weber, M.; Davies, J. J.; Wittig, D.; Oakeley, E. J.; Haase, M.; Lam, W. L.; Schübeler, D. Nat Genet. 2005, 37, 853-862. (18) Taiwo, O.; Wilson, G.A.; Morris, T.; Seisenberger, S.; Reik, W.; Pearce, D.; Beck, S.; Butcher, L.M. Nat Protoc. 2012, 7, 617-636. (19) Bock, C.; Paulsen, M.; Tierling, S.; Mikeska, T.; Lengauer, T.; Walter, J. PLoS Genet. 2006, 2, e26. (20) Bock, C.; Lengauer, T. Bioinformatics 2008, 24, 1-10. (21) Pham, T. T.; Yin, J.; Eid, J. S.; Adams, E.; Lam, R.; Turner, S. W.; Loomis, E. W.; Wang, J. Y.; Hagerman, P. J.; Hanes, J. W. Mol Genet Genomics. 2016, 291, 1491-1504. (22) Zhang, Y.; Liu, T.; Meyer, C. A.; Eeckhoute, J.; Johnson, D. S.; Bernstein, B. E.; Nusbaum, C.; Myers, R. M.; Brown, M.; Li, W.; Liu, X. S. Genome Biol. 2008, 9, R137. (23) Park, P. J. Nat. Rev. Genet. 2009, 10, 669-680. (24) Landt, S. G.; Marinov, G. K.; Kundaje, A.; Kheradpour, P.; Pauli, F.; Batzoglou, S.; Bernstein, B. E.; Bickel, P.; Brown, J. B.; Cayting, P.; Chen, Y.; DeSalvo, G.; Epstein, C.; Fisher-Aylor, K. I.; Euskirchen, G.; Gerstein, M.; Gertz, J.; Hartemink, A. J.; Hoffman,

M. M.; Iyer, V. R.; Jung, Y. L.; Karmakar, S.; Kellis, M.; Kharchenko, P. V.; Li, Q.; Liu, T.; Liu, X. S.; Ma, L.; Milosavljevic, A.; Myers, R. M.; Park, P. J.; Pazin, M. J.; Perry, M. D.; Raha, D.; Reddy, T. E.; Rozowsky, J.; Shoresh, N.; Sidow, A.; Slattery, M.; Stamatoyannopoulos, J. A.; Tolstorukov, M. Y.; White, K. P.; Xi, S.; Farnham, P. J.; Lieb, J. D.; Wold, B. J.; Snyder, M. Genome Res. 2012, 22, 18131831. (25) Szalkowski, A. M.; Schmid, C. D. Brief Bioinform. 2011, 12, 626-633. (26) Yin, R. C., Mao, S. Q., Zhao, B. L., Chong, Z. C., Yang, Y., Zhao, C., Zhang, D. P.; Huang, H.; Gao, J.; Li, Z.; Jiao, Y.; Li, C. P.; Liu, S. Q.; Wu, D. N.; Gu, W. K.; Yang, Y. G.; Xu, G. L.; Wang, H. L. J. Am. Chem. Soc. 2013, 135,10396-10403. (27) Liu, B. D.; Liu, X. L.; Lai, W. Y.; Wang, H. L. Anal Chem. 2017, 89, 6202-6209. (28) Løbner-Olesen, A.; Skovgaard, O.; Marinus, M. G. Curr Opin Microbiol. 2005, 8, 154-160. (29) Song, C. X.; Szulwach, K. E.; Fu, Y.; Dai, Q.; Yi, C. Q.; Li, X. K.; Li, Y. J; Chen, C. H.; Zhang, W.; Jian, X.; Wang, J.; Zhang, L.; Looney, T. J.; Zhang, B. C.; Godley, L. A.; Hicks, L. M.; Lahn, B. T.; Jin, P.; He, C. Nat Biotechnol. 2011, 29, 68-72. (30) Hafner, M.; Landthaler, M.; Burger, L.; Khorshid, M.; Hausser, J.; Berninger, P.; Rothballer, A.; Ascano, M.; Jungkamp, A. C.; Munschauer, M. Cell 2010, 141, 129-141. (31) Chen, K.; Lu, Z. K.; Wang, X.; Fu, Y.; Luo, G. Z.; Liu, N.; Han, D. L.; Dominissini, D.; Dai Q.; Pan, T.; He, C. Angew. Chem. Int. Ed. 2015, 54, 1587-1590. (32) Linder, B.; Grozhik, A. V.; Olarerin-George A. O.; Meydan, C.; Mason, C. E.; Jaffrey, S. R. Nat Methods. 2015, 12, 767-772. (33) Luo, X. T.; Cai, B. D.; Chen, X.; Feng, Y. Q. Anal Chim Acta. 2017, 983, 112-120. (34) Gnerre, S.; Maccallum, I.; Przybylski, D.; Ribeiro, F. J.; Burton, J. N.; Walker, B. J.; Sharpe, T.; Hall, G.; Shea, T. P.; Sykes, S.; Berlin, A. M.; Aird, D.; Costello, M.; Daza, R.; Williams, L.; Nicol, R.; Gnirke, A.; Nusbaum, C.; Lander, E. S.; Jaffe, D. B. Proc Natl Acad Sci 2011, 108, 1513-1518. (35) Yao, B.; Cheng, Y.; Wang, Z.; Li, Y.; Chen, L.; Huang, L. X.; Zhang, W. X.; Chen, D. H.; Wu, H.; Tang, B. S.; Jin, P. Nat Commun. 2017, 8, 1122-1131. (36) Flusberg, B. A.; Webster, D. R.; Lee, J. H.; Travers, K. J.; Olivares, E. C.; Clark, T. A.; Korlach, J.; Turner, S. W. Nat. Methods 2010, 7, 461-465. (37) Fang, G.; Munera, D.; Friedman, D. I.; Mandlik, A.; Chao, M. C.; Banerjee, O.; Feng, Z.; Losic, B.; Mahajan, M. C.; Jabado, O. J.; Deikus, G.; Clark, T. A.; Luong, K.; Murray, I. A.; Davis, B. M.; Keren-Paz, A.;Chess, A.; Roberts, R. J.; Korlach, J.; Turner, S. W.; Kumar, V.; Waldor, M. K.; Schadt, E. E. Nat. Biotechnol. 2012, 30, 1232-1239. (38) Murray, I. A.; Clark, T. A.; Morgan, R. D.; Boitano, M.; Anton, B. P.; Luong, K.; Fomenkov, A.; Turner, S. W.; Korlach, J.; Roberts, R. J. Nucleic Acids Res. 2012, 40, 11450-11462. (39) Wang, Y. Y.; Chen, X.; Sheng, Y. L.; Liu, Y. F.; Gao, S. Nucleic Acids Res. 2017, 45, 11594-11606. (40) Mondo, S. J.; Dannebaum, R. O.; Kuo, R. C.; Louie, K. B.; Bewick, A. J.; LaButti, K.; Haridas, S.; Kuo, A.; Salamov, A.; Ahrendt, S. R.; Lau, R.; Bowen, B. P.; Lipzen, A.; Sullivan, W.; Andreopoulos, B. B.; Clum, A.; Lindquist, E.; Daum, C.; Northen, T. R.; Kunde-Ramamoorthy, G.; Schmitz, R. J.; Gryganskyi, A.; Culley, D.; Magnuson, J.; James, T. Y.; O’Malley, M. A.; Stajich, J. E.; Spatafora, J. W.; Vise, A.; Grigoriev, I. V. Nat Genet. 2017, 49, 964. (41) Jenjaroenpun, P.; Wongsurawat, T.; Pereira, R.; Patumcharoenpol, P.; Ussery, D. W.; Nielsen, J.; Nookaew, I. Nucleic Acids Res. 2018. (42) Luo, G. Z.; Wang, F.; Weng, X.; Chen, K.; Hao, Z.; Yu, M.; Deng, X.; Liu, J.; He, C. Nat Commun. 2016, 7, 11301-11306.



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