BLOOD, 6 DECEMBER 2012. VOLUME 120.

NUMBEFI 24

GWAS SEOUENCE VARIANT FOR PLATELET

VOLUME

4861

PLT-rich plasma (PRP). The PRP was centriluged twice more, each time retaining the supematant and discarding the leukocyte-rich pellet together with the 0.5-mL PRP layer immediately above it. The pRp was further leukol,te-depleted by mixing with anti-CD45 nrlgnetic beads (Dynabeads CD:15, Invitrogen; 33 pL beads/ml of PRP) and rotating at room temperature lbr 20 rninutes. "l'he beads rvere renloveil by Dynal MPC-L magnet, usiug 2 cycles of 2-minute lnagnetization steps and transfer:ring the pRp to fresh tube-s after each step. The leukocyte-deplcted PRP was centrifuged for 10 nrinutes at 15003. The supernatant was discarded and the PLT pellet resuspendeiJ in 2 mL Trizol (Invitrogen) until a plirticle-free solution rvas obtained: l-ml- aliquots were frozen on dr1, ice and stored at -80.C. Startin,' PRP volunres ranged from 5 to 15 mL, ltnd corresponding yields rangecl ' . onr 0.-5 to 2.5 x 10e PUfs.

MK suspension cultures
Mouse f'etal liver cells were collected frorn wr cD1 mice (Charles River Laboratories) on day E13._5 and cultured at 37"C and 5% CO2 in the presence of 0.1 pg/ml purified recombinant mouse c_Mpl ligand fcrr 5 days. Fetal Iivcr cell cultures rvere layered on a single_step gradier.rt (1.5Vc-3.070 BSA) on culture da1,4, and MKs were allowed ro sediment for 30 minutes.2-i The N,lK pellet u,as then resuspcnded in tiesh media and cultured for an additional 24 hours alone or in the presence of either 0.3clc DMSO or 100pM Dl,naSore in DIvISO. NIK cultures were exarninecl on day 5 by phase-contrast microscopv using a Nikon eclipse TSl00 benchtop rnicroscope (Nikon) at 20x magnilicationl digital irrages u,ere collccted on a l{antarnatsu C2400 CICD carrera and analyzcri using IrnageJ. tr'lature N,lKs rvcre identified by size (> lOpm diar-neter) and drsrin,suished from pro-PLT ploducing IVIKs because ol'thc presence or absence of long pro PLT elongations (circularitl > 0.7). Samples were exar.nined in tripli_
cate. and at least 78 cells u,er-e counted for each condition tested.

BNA preparation and cDNA synthesis

cultLrred cells and PLTs was prepared using Trizol essentially according Lo the nianufacturer's instructiorls, except that 2-rnl- Phase I-ock Gel tubes (5') u'cre used for the phase separation. RNA pellets u'ere air dried for no rr)ore than 7 rninutes and theri resuspended in t5 pL nuclease free water (Antbion, Invitrogen). RNA yields averaged 9,10 ng/ l0e PLTS u'ith A260/280 rarios between 1.33 and 1.66. The 25-ng pLT RNA rvlrs processed bl the ABI TaqiVlan Rer,erse Transcription Reagents (Applied Biosl-stems) in a 25-pL volume, accorcling to the manufacturer's protocoi. At the end of the reaction. RNA u,as diluted to 250 pL qith nuclease-free water and stored at -20"C. In addition, total RNA fronr hunan brain (P/N #5.+005, 5:1007. and 5401 17) rvas obrainecl from Stratase're (Agilent Technologies).

ItNA tiom

All studies

complicil uith institutional guicielincs appr.oved by the Children,s Hospital animal carc and use committee, and the lnstitutional Aninral Care and IIse
Committee.

Statistical analysis
Results are expressed as ntcan + SD rvith number of experiments. Statistical cornparisons between groups \rere performed bv 2-tailed I test using Prisnr unlcss stirted olheru.ise. Statistical comparisons of Lucil'erase data rveru perfornred on log transformcd signal intensities using mixed liuear moclels to acc(,Llnt lor tlic hierarchical n:iturc of tlte clata.

Gene expression quantitative real-time PCFI Absolute quxntiiication of Dn'NI3 transcript abunclance in RNA sanrples tl'orn huuran PI-Ts and other cells was carried out on aABI Prism 7900HT Sequencc Dctection Svste-m (Applied Bioslstenrs) using the following prorocol: -50"C I rnrnutes. 9-5oC I0 minutos. 40 x (95"C l5 seconds. 60'C I nrinutc). Ploduct nuntbers of the uscd Tatli\4an gr'nc cxpres\iun assal,s (Appliecl Biosvstems) can be lbunri in suppiemental Tnble l (availeble on the B/o.r.1 Web site; see the Supplemenrai Nlatcrials link at the

Results
MEISI putatively regulated genes are important determinants of the PLT lineage Ther exprcssiot\ ol' MEISI is restr.icted to rhe mesakaryocytic lineagc, anrong dillerentiared blood cclls (supplenrental Iri-surc 1).3 e To urrp N,IEIS i binding cvenrs. we pertbnried Ctrlp-seq in the human urcgakar\ocvtic cell line CHRF. a close rnodel fbr NiKs based on its transcriptional profilc (supplcrnental Figure 2). NIEISI peaks r.r'el'e citlled usiug magnetic-activatecl cell sortingri anJ PICSTe frcnr \\,hich a high-confidence daraset of l3 842 binding events detcoecl bi both algorithnrs \!as serrerated (Figure lA). Gcnes nearbr, NiEIS 1 binding events \\,ere analvzed with GREATTt (Genomic Ilegions Enrichntent of Annotations Tool). revealing that putativeiv reculated genes are significantll, over-represented in

top of the onlino arricle).'lb test lbr abundance of the novel DNMJ
trattscril]t variant. a custonr ThqMan assay was dcsi_sned ri,ith the probe spanninr the boLrndary betrveen the novel exon 28 and e:,cn 3. Transcript lelels rrcrc 1l(rrnr.lized Io Gi\PDH. Reactiorrs uere rncasurecl in tr.iplicate anil prinrt'r el'liciencies obtained through cDNA staudard dilution series. RACE
-5'-Rapid anrpliiication of cDNA ends (RACti) orr RNA sanrples fionr CHRF and \lK cells and PI-Ts rvas pertbrmecl usinc rhe 5'l3'RACE Kir, 2nd Generation (Roche Dia.snostics) fblJouing the manuticturer's protocol. For the PCR artrpliiicarion steps. recombinanr Tecl poll-rncrase and dNTPs tiom Fernteitllis uere used (Fcrnentast. DNA fragmcnts lvere snbclonecl iuto pGent T erisl' (Prornega) and sequenced. Prirner and clone seqlrences can be found in supplemental T.rble 2.

tal

nlegakaryopoiesis and PLT biology,' categor-ies (?rble L; supplemenFi_eure -j). To increase stringency in assigning putativel),

regulated scnes, ii list

r.r'as ser.ieratecl

of

1285 potentialiy N{IJISl-regulared senes

Dual Luciferase reporter assays

DNA ll ti-qrncrrrs u ere clonecl into rhe fire111, Lucitcrlse vector p6i.l. i 0 (Promegrl.'ltn nritlion cells uete transiectetl *.irh I0 p.r lirefll Luciferlse r':clor lrrai -ii]il nS llcnilh L-ueiferase yector pGI-1.7-1 (PlonreglL.l. 'l'ranstecti!)D 1\11s trrrl-rIntttl iirt elerrloooration using a BiLr,liad GencPLriser.\cell
t::1ttittetiti:Lr -:r,,j. ll{) \'. l;tl)lf pF. resisupce u,. -}-itrrrr cu\ctteS). i_riciferrse ae tiviri u as ineasured rn a LUMIstar Optimr iuminonteter (BlvIG l-ubrech r using tirc Dual-I-uciiirase Reporter Assar, kiL (Proniega.l. Random DNA sequenccs lor use ls negative cotltrols \1ere generate(l b1,the Ranclonr DNA Sequc'nce Generator (htqr;11wwu,.facult1'.ucr.edu,/- mnracluro/randont. htnr) and prociucecl b1' artiticia) gene st,nthesis (GencAr.r). I-irr: sequences of

p[otein .o(ilnL {e]tes (sLri)pleinental X13thods). Anall,sis of their. c.\pressir)n Pxttilits slt0ri eil e sigltiticiUtt o\'cr-rcprescntl,rion ol -i7 q*nes ol il sct ol'l-jl tltat $cre sh()\\'it to he sfrnn-ulr,()\.1-r.L-.{Dr.c,ssdJ
(P < -5 x l0-rrt Figure lBl in the HaernAtlas colnpcndium of biood ceil trtnscripts.s Thcse obser.r,ations are coltsistent with our ll,pethcsis that t'1El.91, hc:cause of'its character-istic finclge resn.i.tion, r'esulates rnirnv gcnes critical to the identitv ol N{Ks and pLTs. Incleed. thesc -57 gcnes contain ntan\, \\,ell-known resulators of their firnction. such rs the coilrrgen siqnaling rcceptor Glycr)protein (GP) \rl ((i['rtl. 1 of the .tr suhLurits oi'tlre PLT recepror for vou \\iillchrancl liactor. GPV iG'P-i1. and rhe r qranule rnctnbrene protei n P-sclectin (.lfLP).

hv filtcring firr pelks \\'ith a rninirrunl height oi' 30 reuris (S()tir ircrrcntile oi peak hei-ithil ancl r.vithin - -i kb o1,

in \lK. r'iiilti\'3 tr) '.itJ i)thar -

tntrttu-i bloocl

eil

l_t

lc:,

cloninl prinrers anil ccinstruct inserts.

sec supplernentiLl Table 3.

Electrophoretic mobility shift assay

See srrl-.plentcntrLl lvlcthorls.