Capture-CofHoxa1inMusmusculus:WTrep3
Cellswerefixedwith1%(vol/vol)formaldehydefor10minatroomtemperature,quenchedwith125mMglycineinPBS,andthenlysedincoldlysisbuffer(10mMTrisHCl,pH7.5,10mMNaCl,5mMMgCl2,0.1mMEGTA,0.2%NP-40,1completeproteaseinhibitorcocktail(Roche)).ChromatinwasdigestedwithDpnII(NewEnglandBiolabs)at37Covernight.FragmentswerethendilutedandligatedwithT4DNAligase(Takara)at16Covernight.Cross-linkingwasreversedbyovernightincubationat60CwithproteinaseK(Bioline).Then3Clibrarieswerepurifiedbyphenol-chloroformfollowedbychloroformextractionandethanol-precipitatedat80Covernight.Sequencinglibrarieswerepreparedfrom10gofthe3Clibrarybysonicationtoanaveragesizeof200~300bpandindexedusingNEBnextreagents(NewEnglandBiolabs),accordingtothemanufacturer'sprotocol.Enrichmentof2gofanindexedlibraryincubatedwith3uMofapoolofbiotinylatedoligonucleotides(probesequencesarelistedinSupplementaryTable7)wasperformedusingtheSeqCapEZHybridizationreagentkit(#05634261001,Roche/NimbleGen),followingthemanufacturer'sinstructions.Tworoundsofcaptureemploying48~72and24hrhybridizations,respectively,wereused.Correctlibrarysizewasconfirmedbyagarosegelelectrophoresis,andDNAconcentrationsweredeterminedusingaQubit2.0Fluorometer(ThermoFisherScientific).AllsequencingwasperformedonHi-Seq2500platformsusingpaired150bpprotocols(Illumina).