ChIPresultsobtainedwiththeantibodydirectedagainstH3K56ac
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K56ac(Cat.No.25259)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1.5millioncells.Atitrationoftheantibodyconsistingof0.5,1,2and,5μgperChIPexperimentwasanalysed.IgG(1μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforaregionapproximately1kbupstreamoftheGAPDHpromoterandfortheEIF4A2promoter,usedaspositivecontrols,andforthecodingregionoftheinactiveMYOD1geneandtheSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K56ac
ChIPwasperformedonshearedchromatinfrom1.5millionHeLaS3cellsusing5μgoftheantibodyagainstH3K56ac(cat.No.25259)asdescribedabove.TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaHiSeq.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The51bptagswerealignedtothehumangenomeusingtheBWAalgorithm.Figure2showstheenrichmentalongthecompletesequenceanda1MbregionoftheX-chromosome(fig2AandB)andingenomicregionsofchromosome12and3,surroundingtheGAPDHandEIF4A2positivecontrolgenes.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K56ac(Cat.No.25259)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:15,300.

CrossreactivitytestsusingtheantibodydirectedagainstH3K56ac
TotestthecrossreactivityoftheantibodyagainstH3K56ac(Cat.No.25259),aDotBlotanalysiswasperformedwithpeptidescontainingotherhistonemodificationsandtheunmodifiedH3K56.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

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