ProcedureGrindinmortarandpestleorWaringblenderwith5-7volumesbufferApergtissue.UseMCEat350l/L,andifnecessary,with5ml1MDIECA/L.Squeezethroughcheesecloth,twolayersofMiracloth.10minat1000gDecantsupernatantandcentrifuge10minat15,900g.ResUSPendeachpelletinafewdropsofbufferGwithpaintbrush;combine;bringtoabout10ml/50g,15ml/75g.10minat1000g;pouroffmost;swirlpellettoremovefluffylayer;combine.Bringsupernatantto10mMMgCl2(100l1M/10ml).Bringto20gDNase/ml(100l2mg/ml/10ml).60min.4C.Underlayshelfbuffer,20ml/10-15ml;alwaysuse20mlormore.20minat12000g.Resuspendinsmallvolumeshelfbufferwithbrush;bringtoabout10ml/50-100g.10minat15900g.ResuspendpelletsinNN(lysis)buffer(4-5ml/50-75g).AddSDSto0.5%(250lof10%/5mlNN).Swirlthoroughly.AddproteinaseKto100g/ml(25lof20mg/ml/5mlNN).Swirlgently.60min.37C.Addequalvolumeof3:1water-saturatedphenol,chloroform-isoamylalcoholmixture.Emulsifyca.5min.10minat7000g.Collectsupernatant;repeat17and18:3totalextractions.Finalsupernatant;add0.1volume8MAmmoniumacetate;thenadd2volumesofabsoluteethanol.60min,-80C;10minat8000-9000g;drain;addequalvolume70%ethanol;letsit10min;10minat8000-9000g;draindry.Vacuumdrypellet,30min.Twosmallcorextubesarebetterthanone30mlCorex.Add100-500l0.1XNTE,10lRNasemixture.Typicallyuse500lper50gtissue.Hydrate30min.,37C.