ChIPresultsobtainedwiththeantibodydirectedagainstH3K27me3
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K27me3(Cat.No.25244)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5,and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromotersoftheactivegenesEIF4A2andGAPDHasnegativecontrols,andforthecodingregionsoftheinactivegenesMYT1andTSH2Baspositivecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthatH3K27me3ispreferablypresentatinactivegenes.

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K27me3
ChIPwasperformedonshearedchromatinfrom1millionHeLaS3cellsusing1μgoftheantibodyagainstH3K27me3(Cat.No.25244)asdescribedabove.TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaHiSeq.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The51bptagswerealignedtothehumangenomeusingtheBWAalgorithm.Figure2showstheenrichmentingenomicregionsofchromosome6,surroundingtheTSH2Bgene(indicatedbyanarrow;fig2A),ofchromosome20,surroundingtheMYT1gene(fig2B),andofchromosome2and3(figure2CandD).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K27me3(Cat.No.25244).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:3,500.

CrossreactivitytestoftheantibodydirectedagainstH3K27me3
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K27me3(Cat.No.25244)withpeptidescontainingothermodificationsofhistoneH3andH4andtheunmodifiedH3K27sequence.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:5,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.PleasenotethatthatantibodyalsorecognizesthemodificationifS28isphosphorylated.

WesternblotanalysisusingtheantibodydirectedagainstH3K27me3
Westernblotwasperformedonwholecell(25μg,lane1)andhistoneextracts(15μg,lane2)fromHeLacells,andon1μgofrecombinanthistoneH2A,H2B,H3andH4(lane3,4,5and6,respectively)usingtheantibodyagainstH3K27me3(Cat.No.25244)diluted1:500inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K27me3
MouseNIH3T3cellswerestainedwiththeantibodyagainstH3K27me3(Cat.No.25244)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K27me3antibody(left)diluted1:200inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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