MeDIP-seq(methylatedDNAimmunoprecipitation-sequencing)withthemonoclonalantibodydirectedagainst5-mC
GenomicDNAfromE14EScellswasshearedtogeneraterandomfragments(sizerange300to700bp).OneμgofthefragmentedDNAwasligatedtoIlluminaadaptersandtheresultingDNAwasusedforastandardMeDIPassay,using2μgofthemonoclonalagainst5-mC(Cat.#25206).AfterrecoveryofthemethylatedDNA,IlluminasequencinglibrariesweregeneratedandsequencedonanIlluminaGenomeAnalyzeraccordingtothemanufacturer’sinstructions.Figure1Aand1BshowGenomebrowserviewsofCAsimplerepeatelementswithreaddistributionsspecificfor5-mCat2genelocations(SigleC15andMfsd4).VisualinspectionofthepeakprofilesinagenomebrowserrevealshighenrichmentofCAsimplerepeatsinaffinity-enrichedmethylatedfragmentsafterMeDIPwiththe5-mCmonoclonalantibody.

MeDIPresultsobtainedwiththemonoclonalantibodydirectedagainst5-mC
MeDIP(MethylatedDNAimmunoprecipitation)wasperformedonfragmentedhumangenomicDNAusingthemonoclonalantibodyagainst5-mC(Cat.#25206).ThefragmentedDNAwasspikedwiththeinternalcontrolspresentinthekit(methylatedDNA(meDNA)asapositiveandunmethylatedDNA(unDNA)asanegativecontrol)priortoperformingtheIP.QPCRwasperformedwithoptimizedprimersets,includedinthekit,specificforthemethylatedandunmethylatedDNAcontrols,andforaknownmethylated(TSH2B)andunmethylated(GAPDH)genomicregion.Anadditionalinternalpositiveandnegativecontrollocus(4994+and8804-,respectively)werealsotested(4994+:forwardprimer5’-GGGAATATAAGGAGCGCACA-3’andreverseprimer5’-TCGGTTAAAACGGTCAGGTC-3’;8804-:forwardprimer5’-CGAGGCGTGAGTTATTCCTG-3’andreverseprimer5’-CTCTTGTGGCTGAGCTCCTT-3’).Figure2showstherecovery(meanof3experiments),expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ELISAusingthemonoclonalantibodydirectedagainst5-mC
ELISAwasperformedusingmonoclonalantibodyagainst5-mC(Cat.#25206),diluted1:100.Thewellswerecoatedwithaserialdilutionofhydroxymethylated(hmC),methylated(mC)andunmethylated(C)DNAstandards.

Dotblotanalysisusingthemonoclonalantibodydirectedagainst5-mC
Todemonstratethespecificityoftheantibodyagainst5-mC(Cat.#25206),aDotblotanalysiswasperformedusinghydroxymethylated(hmC),methylated(mC)andunmethylated(C)DNAstandards.Onehundredto4ng(equivalentof5to0.2pmolofC-bases)ofthecontrolswerespottedonamembrane.Theantibodywasusedatadilutionof1:250.Figure3showsahighspecificityoftheantibodyforthemethylatedcontrol.

Immunofluorescenceresultsobtainedwiththemonoclonalantibodydirectedagainst5-mC
Figure5A:HumanHeLacellswerestainedwiththemonoclonalantibodyagainst5-mC(Cat.#25206).Cellswerefixedwith4%formaldehydeinPBSfor10min.atroomtemperature,permeABIlisedwith0.5%TritonX-100for1hourandtreatedwith2NHClfor1hour.AfterblockingwithPBScontaining0.1%TritonX-100and1%BSA,thecellswereimmunofluorescentlylabeledwiththe5-mCantibody(left)diluted1:500inblockingsolution,followedbyagoatanti-mouseantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.
Figure5B:ImmunofluorescentstainingofaninterphaseHeLacellwiththe5-mCantibodyfollowedbyagoatanti-mouseantibodyconjugatedtoFITC(yellow)andwithHoechststaining(blue).

Surfaceplasmonresonance(SPR)analysisofthethemonoclonalantibodydirectedagainst5-mC
Asynthesizedbiotin-labeled5-mCconjugatewasimmobilizedonaCM4BIAcoresensorchip(GEHealthcare,France).Briefly,twoflowcellswerepreparedbysequentialinjectionsofEDC/NHS,streptavidin,andethanolamine.Oneoftheseflowcellsservedasnegativecontrol(biotinylatedspacerwithout5-mC),whilebiotinylated5-mCconjugatewasinjectedintheotherone,toobtainanimmobilizationlevelof55responseunits(RU).AllSPRexperimentswereperformedusingHBS-Nbuffer(10mMHEPES,150mMNaCl,pH7.4)ataflowrateof5μl/min.Interactionassaysinvolvedinjectionsof3differentdilutionsofthe5-mCmonoclonalantibody(Cat.#25206)overthebiotinylated5-mCconjugateandnegativecontrolsurfaces,followedbya3minwashingstepwithHBS-Nbuffertoallowdissociationofthecomplexesformed.Attheendofeachcycle,thestreptavidinsurfacewasregeneratedbyinjectionof0.1Mcitricacid,pH3.Thesensorgramscorrespondtothebiotinylated5-mCconjugatesurfacesignalsubtractedwiththenegativecontrol.DatafromthesensorgramsthatreachedbindingequilibriumwereusedforScatchardanalysis.Thevalueofthedissociationconstant(kd)obtainedbyglobalfittingand1:1Langmuirmodelis13±9nM.

Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.