Cell Stem Cell

Supplemental Information

Combined Single-Cell Functional

and Gene Expression Analysis Resolves

Heterogeneity within Stem Cell Populations

Nicola K. Wilson, David G. Kent, Florian Buettner, Mona Shehata, Iain C. Macaulay,

Fernando J. Calero-Nieto, Manuel Sánchez Castillo, Caroline A. Oedekoven, Evangelia

Diamanti, Reiner Schulte, Chris P. Ponting, Thierry Voet, Carlos Caldas, John Stingl,

Anthony R. Green, Fabian J. Theis, and Berthold Göttgens

B

% P

urity

by

func

tiona

l si

ngle

cel

l ass

ay

C C

him

eris

m (%

) (D

onor

/Don

or+R

ecip

ient

)

LSK CD34

SLAM LSK

SPKLS 150

Line

age

cont

ribut

ion

to d

onor

cl

one

(%)

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

LSK CD34 SLAM LSK SP KLS 150

D

A

CD45

EPCR EPCR

CD48

CD150

CD45

EPCR

HSC2 & HSC5

HSC5 HSC2

CD45

c-KI

T

Sca1

FLK2

CD48

CD15

0

CD34

HSC1 c-KI

T

Sca1

HSC1

c-KI

T

Sca1

c-KI

T

FLK2

CD34

HSC3 HSC3

Sca1 c-

KIT

Sca1

Hoec

hst b

lue

Hoechst red

Hoec

hst b

lue

Hoechst red

CD15

0

FSC

HSC4

HSC4

Wilson et al Figure S1

−100 −50 0 50 100−80

−60

−40

−20

0

20

40

60

80

100

−100 −50 0 50 100−80

−60

−40

−20

0

20

40

60

80

100

A B

−50

0

50

−50 0 50 100

−50

0

50

−50 0 50 100

−50

0

50

−50 0 50 100

−50

0

50

−50 0 50 100

−50

0

50

−50 0 50 100

−50

0

50

−50 0 50 100

−50

0

50

−50 0 50 100

−50

0

50

−50 0 50 100

HSC1 HSC2 HSC3

HSC4 HSC5 LMPP

MEPGMP

C

−0.2 0 0.2

7AAD

CD150**

CD229

CD34

CD45

CD48*

EPCR

FSC

Flt3

Hoechst Red

Hoechst blue

Lineage neg

SSC

ScaI

c−kit

Norm. marker expression−0.1 0 0.1

FSC

SSC

CD48

CD229

EPCR

7AAD

CD45

CD150

HSC2

−0.1 0 0.1

FSC

SSC

CD34

CD229

c−kit

Flt3

7AAD

ScaI**

Lineage neg

HSC3

−0.2 0 0.2

FSC

SSC

CD34

7AAD

CD48

c−kit

Flt3

CD150**

ScaI**

Lineage neg

HSC1

−0.1 0 0.1

FSC

SSC

7AAD

CD229

c−kit

ScaI

CD150

Lineage neg

Hoechst blue

Hoechst Red

HSC4

−0.1 0 0.1

FSC

SSC

CD45

CD150

CD48

EPCR

7AAD

HSC5

Norm. marker expression Norm. marker expression Norm. marker expression Norm. marker expression Norm. marker expression

D

1 10 100 1000 100001

10

100

1000

10000

Sca1

c−ki

t

1 10 100 1000 100001

10

100

1000

10000

Sca1

c−ki

t

1 10 100 1000 100001

10

100

1000

10000

CD150

CD

48

1 10 100 1000 100000

10000

20000

30000

40000

50000

CD150

SS

C

1 10 100 1000 10000

1

10

100

1000

10000

CD150

CD

48

1 10 100 1000 10000

1

10

100

1000

10000

Sca1

c−ki

t

1 10 100 1000 100000

10000

20000

30000

40000

50000

CD150

SS

C

1 10 100 1000 100001

10

100

1000

10000

CD150

CD

48

1 10 100 1000 100000

10000

20000

30000

40000

50000

CD150

SS

C

1 10 100 1000 100000

10000

20000

30000

40000

50000

CD150

SS

C

HSC1 HSC2 HSC3 HSC4 HSC5

c-ki

t vs

Sca

1C

D48

vs

CD

150

SS

C v

s C

D15

0

E MolONoMO

N/A

N/A

N/A

N/A

N/A

Wilson et al Figure S2

−50

0

50

−50 0 50 100

CMP

−0.08 −0.06 −0.04 −0.02 0 0.02 0.04 0.06 0.08

−0.08

−0.06

−0.04

−0.02

0

0.02

0.04

0.06

0.08

0610010F05Rik0610012H03Rik

0610031J06Rik

0610040J01Rik

1110008J03Rik

1110008L16Rik1110018G07Rik

1110034G24Rik

1110051M20Rik

1110059E24Rik

1110059G10Rik1190002N15Rik

1200007C13Rik

1200011I18Rik

1200014J11Rik

1500009L16Rik

1600012H06Rik1600014C10Rik

1700012B15Rik

1700017B05Rik

1700019D03Rik

1700020I14Rik1700029J07Rik

1700030K09Rik

1700037H04Rik

1700048O20Rik

1700049G17Rik

1700052N19Rik1700066M21Rik

1700086P04Rik

1810011H11Rik

1810024B03Rik

1810026J23Rik1810055G02Rik

1810062G17Rik

2010012P19Rik

2010015L04Rik

2010315B03Rik

2210018M11Rik

2210404O09Rik

2210408I21Rik

2310005G13Rik

2310008H04Rik2310035C23Rik

2310044G17Rik

2410003L11Rik2410004P03Rik2410018L13Rik2410089E03Rik

2410131K14Rik

2510003E04Rik2510009E07Rik

2610002M06Rik

2610008E11Rik

2610015P09Rik

2610018G03Rik2610019F03Rik

2610020H08Rik

2610021A01Rik

2610034B18Rik2610301B20Rik2610301G19Rik2610305D13Rik

2610307P16Rik

2610318N02Rik2700049A03Rik

2700081O15Rik

2700097O09Rik

2810021J22Rik

2810055G20Rik

2810403D21Rik

2810408A11Rik2810417H13Rik

2810474O19Rik

2900005J15Rik2900011O08Rik3110001I22Rik3110040N11Rik

3110043O21Rik

3110052M02Rik

3110067C02Rik

3300002I08Rik

3632451O06Rik

4632427E13Rik

4632428N05Rik

4833418N02Rik

4833439L19Rik

4921507P07Rik4921531C22Rik

4930402H24Rik

4930404I05Rik

4930412C18Rik

4930412M03Rik

4930422G04Rik4930447C04Rik4930451G09Rik

4930515G01Rik4930522L14Rik

4930562F07Rik

4930594C11Rik

4930596I21Rik

4931406C07Rik4933406P04Rik

4933411K20Rik4933424M12Rik

4933426M11Rik

4933436C20Rik5031439G07Rik

5330417C22Rik

5430403G16Rik

5430427O19Rik

5530601H04Rik

5730455P16Rik

5830418K08Rik5830428H23Rik5830444B04Rik

6330408A02Rik6330549D23Rik8430408G22Rik

8430419K02Rik

8430419L09Rik

8430429K09Rik

8430432A02Rik

9030617O03Rik

9030619P08Rik

9130008F23Rik9130019O22Rik

9330132A10Rik

9330182L06Rik

9430008C03Rik

9430015G10Rik

9430020K01Rik

9430023L20Rik9530051G07Rik

9530068E07Rik9530077C05Rik

9530080O11Rik

9630013D21Rik

9830147E19Rik

9930021J03Rik9930104L06RikA030009H04Rik

A230046K03Rik

A330021E22Rik

A430033K04Rik

A430078G23Rik

A430078I02Rik

A530054K11Rik

A630007B06RikA630033H20Rik

A630052C17Rik

A830080D01Rik

AA386476AA987161

AC087901.1

AC102484.1AC102739.1

AC107704.1AC107846.1AC113309.1

AC113320.1AC114820.1

AC117574.1AC119212.1AC121121.1AC121499.1

AC121821.1

AC122371.1

AC122487.1

AC123687.1AC123702.1AC123702.2

AC124322.1AC125223.1

AC125399.1AC127314.1

AC134826.1AC136008.1AC137938.1

AC137970.1

AC140312.1

AC142191.1AC149090.1AC151841.1

AC153506.1AC154235.1AC154274.1AC154486.1

AC158296.1AC159256.1AC161602.1

AC162936.1

AC163677.1AC164431.1AC165246.1

AC165259.1AC166827.1AC167018.1AC168090.1AI182371

AI504432AI597468

AI597479AI848285

AI854703

AI987944

AK129341

AL663030.1AL844859.1

AU041133

AW112010

AW146154

AW209491

AW549877AW551984

Aaas

Aacs

Aagab

Aars

Aasdhppt

Abca1

Abca4

Abcb7

Abcb8

Abcc1

Abcd1 Abcd3 Abce1

Abcg1

Abcg3

Abhd13Abhd14bAbhd17bAbhd4

Abi1

Abi2

Abl2

Ablim1

Ablim2Ablim3

Abr

Abtb1Abtb2

Acad9

Acadl

Acadsb

Acap1

Acat2Acbd3

Acbd5Acer2

Acer3

Ache

Acn9

Acot1Acot9

Acox2

Acox3Acp5Acpl2

Acpp

Acsbg1

Acsl3

Acsl4

Acss1

Acss2

Acss3

Actn4

Actr3

Actr3b

Actr6

Actr8

Acy3

AdaAdal

Adam10

Adam22

Adam5Adam9

Adamts10

Adamts6

Adap1Adar

Adat2

Adck1

Adck3

Adcy7

Add3

AdkAdo

Adora2bAdprh

Adprm

Adss

Aebp2A�1

Afg3l1

Aftph

Aga

Agap2

Agap3

Agfg1AglAgo1

Ago2Ago4

Agpat2

Agpat6

Agphd1AgrnAgtpbp1

Agtr1a

Agxt2l2

Ahctf1Ahnak

AhrAhrrAifm2

Aig1

Aire

AirnAjuba

Ak1

Ak3Ak4

Akap11

Akap7

Akap8

Akirin1

Akr1c19

Aktip

Alcam Aldh16a1

Aldh1a1Aldh1b1

Aldh3a2

Aldh6a1

Aldh9a1

Aldoc

Alg10b

Alg11

Alg13

Alg2

Alg3

Alg6

Alg9

Alox15

Alpk3

Als2cl

AmfrAmigo2

Ammecr1

Amn1

Amot

Amz2

Anapc1Anapc2

Anapc4Anapc5

Angel1

Angptl2

Ank

Ankef1

Ankfy1

Ankhd1

Ankra2

Ankrd10

Ankrd16Ankrd17

Ankrd24

Ankrd26

Ankrd28

Ankrd32

Ankrd33b

Ankrd39

Ankrd42

Ankrd49

Ankrd50Ankrd52

Anks1Ano10

Antxr2

Anxa1

Anxa3

Anxa4

Anxa6

Anxa7Ap1g1

Ap1g2Ap1s2

Ap3m2

Ap3s1

Ap4b1

Ap5b1

Ap5m1

Apbb1

ApcAplf

AplnAplp2

Apob

Apobec1

Apoe

Apol9b

Apoo

AppAppl2

Aptx Aqp1

Aqp11Aqp9

Ar

Araf

Arap2

Arf4

Arfgef1

Ar�p1

Arhgap12

Arhgap15

Arhgap18

Arhgap26Arhgap27

Arhgap28

Arhgap31

Arhgap5

Arhgef25

Arhgef28Arhgef6

Arhgef9

Arid1b

Arid2

Arid3b

Arid4b

Arid5b

Arih1Arl11Arl5b

Arl6Arl6ip6Arl8bArmcx1Armcx4

Armcx6

Arntl

Arpp19

Arrdc3

Arsa

Arsk

Art4

Art5

Arxes2

As3mtAsah1

Asah2Asap1Asb1

Asb13

Asb4

Asb6

Asb7

Ascc2

Ascc3

Ascl2Asf1b

Ash2l

Asnsd1

Aspa

AsphAspm

Aste1

Asun

Atad3a

Atad5

Atf1

Atf2

Atf7ip

Atg10

Atg12

Atg14Atg16l1

Atg16l2

Atg2a

Atg3

Atg4a

Atg5Athl1

Atl2

Atm

Atmin

Atp11a

Atp11c

Atp13a1

Atp13a2

Atp1b3

Atp2a2

Atp2b4

Atp2c2Atp5sAtp6ap2

Atp6v0a1

Atp6v0a2

Atp6v1a

Atp6v1g2Atp7a

Atp7bAtp9b

Atpbd4

Atr

AtrxAtxn1l

Atxn7

Atxn7l2AurkaAurkb

Avl9

Awat2Axl

Azi2

Azin1

B130034C11Rik

B230120H23Rik

B230217C12RikB230219D22RikB230307C23Rik

B230378P21Rik

B3galnt1

B3galt6

B3gnt3

B3gnt5

B3gnt7

B4galnt1B4galt4

B630005N14RikB630019K06Rik

B930095G15Rik

BC003331

BC005561

BC005764

BC016423

BC017643BC018507

BC023829

BC026585

BC030307

BC030336BC043934 BC052040

Bace2

Bach1

Baiap2

BambiBambi−ps1Bank1

Banp

Bap1

Batf2

Baz2b

Bbs10Bbs12

Bbs4

Bbs5

BbxBcam

Bcap29Bcar3Bcas3

Bckdk

Bcl11a Bcl2Bcl2l14

Bcl6Bcl6b

Bcl9

Bclaf1

Bco2 Bcor

Bdh1

Bdh2

Bdp1

Bend7Best1

Bet1Bet1l

BgnBhlha15

Bhlhb9

Bhlhe40Bicd1

Bicd2

Birc2

Birc5

Birc6

Bivm

Bloc1s6

Blzf1

Bmf

Bmi1

Bmp4 Bmp8a

Bmpr1aBmx

Bnip3

Bod1l Bop1

Bora

Bpifb5

BpifcBpnt1

BptfBraf

Brca2Brd7Brms1lBrwd1

Bsdc1Btaf1

Btbd10

Btbd19Btbd6

Btbd9

Btd

Btg1Btg2

Bub1b

Bysl

Bzw2

C030039L03RikC130050O18Rik

C1d

C1galt1C1galt1c1

C1ra

C1rl

C230062I16RikC230081A13Rik

C2cd3

C330007P06RikC330018D20Rik

C330027C09Rik

C530008M17Rik

C78339CAAA01201205.1

CT572998.1CT573100.1

Cab39

Cabp4 Cacfd1Cacna2d4

Cacul1

Cacybp

Cad

Cadm1

Cadps2

Calcrl

Calhm2

Calml4

Camk1d

Camk2d

Camk2n1

Camk4

Camkk1

Camsap1CanxCapn2

Capn3

Capn5

Capn7Caprin1

Caps2 Car13Car2 Car3

Car5bCar8

Car9Card10

Casc4

Casc5

Casd1

Cask

Casp1 Casp12

Casp2

Casp4

Casp7

Casp8ap2

Casp9 Cast

Cat

Catsperd

Cav2

Cbfa2t2

Cbfb

Cbll1

Cbx4

Cbx7

Ccar1

Ccbl1

Ccbl2

Ccdc114

Ccdc130

Ccdc132

Ccdc136Ccdc141

Ccdc149

Ccdc155Ccdc157

Ccdc17

Ccdc18

Ccdc25

Ccdc47

Ccdc50Ccdc57Ccdc62

Ccdc64Ccdc66

Ccdc71l

Ccdc88a

Ccl3Ccl4

Ccl9

Ccna2

Ccnb1Ccnb2

Ccnb2−psCcnc

Ccne2Ccnf

Ccng1

Ccng2

CcnjCcnjl

Ccnl1

Ccnt1

Ccrl2

Ccrn4l

Ccser2

Cd151

Cd164

Cd1d1

Cd200r1

Cd27

Cd28

Cd2ap

Cd302

Cd34

Cd36

Cd37

Cd38

Cd4

Cd44

Cd53

Cd55Cd68

Cd69

Cd74

Cd82Cd83

Cd84 Cd86

Cd96

Cd99l2

Cdc14a

Cdc14b Cdc16

Cdc20

Cdc25bCdc25c

Cdc27

Cdc40

Cdc42ep4

Cdc42se2Cdc45Cdc6

Cdc73

Cdca2

Cdca3

Cdca5Cdca7

Cdca8

Cdh23

Cdk1Cdk11b

Cdk18

Cdk19Cdk2

Cdk5rap1

Cdk5rap2

Cdk6

Cdk8Cdkn1b

Cdkn1c

Cdkn2aipCdkn2aipnlCdkn3

Cdpf1Cds1

Cdt1

Cdyl2Celf2

Cenpa

Cenpc1

Cenpe

Cenpf

CenphCenpi

Cenpk

Cenpl

Cenpo

Cep110

Cep135

Cep152

Cep290Cep41

Cep55

Cep57

Cep57l1

Cept1

CerkCers5

Cers6Ces2g

Cetn3

C�2

Cggbp1

Chac2

Chad

Chaf1aChamp1

Chd3 Chd9Chek1

Chi3l7

Chic1

Chid1Chka

Chm

Chmp2b

Chmp5

Chn2

Chordc1

Chp1

Chpt1

Chrm1

Chst10

Chst11

Chst14Chst7 Chtop

Chuk

Cisd2

Cish

Cited2

Ckap2

Ckap2lCkap4

Cks1bCks2

Clca1

Clcf1 Clcn3Clcn5Clcn6

Clcn7Cldn10

Cldn12Cldn4

Cldn5

Cldn9

Clec1aClec1b

Clec4e

Clec7a

Clic6

Clip3

Clk1

Clk4

Cln5

Clock

Clp1

Clpb

Clptm1lClstn3

Cltc

Clu

Cluh

Cmas

Cmklr1Cmpk1

Cmpk2Cmtm6

Cmtm7

Cndp2

Cnksr1Cnn3

Cnnm3

Cnot10Cnot2

Cnot6

Cnot6l

Cnot7Coa5

Coasy

Cog6

Col10a1

Col18a1Col4a1 Cope

Copg2Cops2Coq10bCoq3

Coq4Coq6

Coro7

Cpd

Cpeb2Cped1Cpne3

Cpne8Cpq

Cpsf2

Cpsf3

Cpsf6Cpt1c

Cpt2

Cpxm1Cr2

Crat

Crbn

Creb1Creb5

Crebrf

Crebzf

Creld1

Cript

Crispld1

Crk

Crnkl1

Crot

Cryl1

Cryz

Csad

Csf2rb

Csf2rb2

Csgalnact1 Csgalnact2

Csnk1g1

Csnk1g2Csnk1g3Cspp1

Csrp2bp

Cstf1

Cstf2

Cstf2t

Cstf3Ctbs

Ctdsp2

Ctdspl

Ctdspl2

Ctla2a

Ctla2b

Ctnnal1

Ctps

Ctps2

Ctsc

CtsfCtsh

Ctsl

Ctso

Ctss Ctsw

Cttn

Ctu1

Cul2

Cul3Cul4a

Cul4b

Cul5

Cul9Cx3cl1

Cyb561a3Cyb5d2

Cyb5r1

Cyb5r3

Cy�p1

Cyld

Cyp20a1

Cyp26b1

Cyp27a1

Cyp2j6Cyp2j9 Cyp2r1Cyp39a1

Cyp4b1

Cyp4f13Cyp4v3

Cyp51

Cyr61Cysltr2

Cyth3

Cyth4

CytipD10Wsu52e

D11Wsu47e

D130040H23Rik

D14Abb1e

D15Ertd621e

D16Ertd472e

D17H6S56E−5

D17Wsu92e

D230025D16RikD3Ertd254e

D3Ertd751e

D4Wsu53eD630039A03RikD630045J12Rik

D6Wsu163e

D930028M14RikD930048N14Rik

Dach1Dag1

DaglbDapk2

Dars2Dazl Dbt

Dcaf10

Dcaf12

Dcaf12l1

Dcaf12l2

Dcaf15

Dcaf7

Dcbld1Dcdc2b

DckDclre1a

DctdDcun1d2

Dcun1d4Ddah2

Ddb2

Ddit3

Ddit4l

DdostDdx1

Ddx20Ddx28Ddx31

Ddx3xDdx4 Ddx50 Ddx52

Ddx58

Ddx59Ddx6

Ddx60

Def8

Degs1

Dek

Dennd1b

Dennd1c

Dennd2d

Dennd4a

Dennd6aDepdc1aDepdc7

Deptor

Dfna5

Dgat1Dgcr14

DgkaDgkq

Dhcr7

Dhfr

Dhodh

Dhrs1

Dhrs13

Dhrs3

Dhrs7 Dhx15Dhx16

Dhx33

Dhx35Dhx36

Dhx40Dhx58Diap2

Dis3l2 Dkc1

Dkkl1

Dld

Dlg2Dlg3

Dlgap5Dll4Dlst

Dmrtc1a

Dmtf1

DmwdDmxl1

Dmxl2

Dnahc1

Dnaja1Dnaja2

Dnajb14

Dnajb4Dnajb9

Dnajc10Dnajc13

Dnajc14

Dnajc21

Dnajc27

Dnajc28

Dnajc9

Dnase1l1Dnase2aDnm2

Dnm3

Dnmt1Dnmt3aDnph1

Dock10

Dock3

Dock5

Dock6Dock9

Dok2Dok3

DolkDpp3

Dpp4

Dpp8

Dpy19l1Dpy19l3

Dr1

Dram2

Drg2Dscc1Dscr3Dse

Dsel

Dsg2

Dsn1

Dtnbp1

Dtx2

Dtx3l

Dus1l

Dusp1

Dusp10Dusp18

Dusp2

Dusp26Dusp4 Dusp6Dync1i1

Dync1li2

Dync2li1

Dynll1Dynlt3

Dyrk3

Dzip3

E030002O03Rik

E130018N17Rik

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Pigk

Pigv Pik3c2a

Pik3c3 Pik3caPik3cg

Pik3ip1

Pik3r1

Pik3r2Pik3r5

Pim2Pink1

Pip5k1b

Pir Pitpna

Pitpnb

Pitpnc1

Pitx2

Piwil4

Pja1

Pkd1

Pkd2

Pkhd1l1

Pkia

Pkn3

Pknox1Pkp3

Pkp4

Pla2g15

Pla2g4c

Pla2g7

PlaaPlac8

Plag1

Plagl1

Plagl2

Plbd1

Plbd2

Plcb1

Plcb4

Plcl1

Plcl2

Pld3

Pld4

Pld6 Plek

Plekhb1

Plekhf1

Plekho2

Plk1

Plk1s1

Plk2

PlnPlod1Plp1Plrg1

Pls3

Plscr2

Plscr4

Plxdc2Plxna2

Plxnb2

Plxnc1

Pm20d1Pml

Pmm1Pmm2

Pmp22

PmpcaPmpcb

Pms1

Pms2Pnck

Pnldc1

Pnpla7

Pnpla8

Pnpt1

Pnrc1Pnrc2 Poc1a

Pofut1

Pofut2

Pogk

Pogz

Pola1

Pold1Pold2

Pole2

Polk

PollPolm

Polr2b

Polr3e

Polr3gPolr3h

Polr3k

Polrmt Pomt1

Pon3

Pop1

Porcn PostnPot1a

Pou2f1

Pou5f2

Ppa1Ppa2Ppap2b

Ppap2cPpapdc1b

Ppapdc2

Ppapdc3

Ppargc1a

Ppat

Ppcdc

Pp�bp1Pp�bp2

Ppif

Ppil4

Ppip5k2

Ppm1aPpm1b

Ppm1d Ppm1fPpm1g

Ppm1l

Ppme1

Ppox

Ppp1cb

Ppp1r12bPpp1r15aPpp1r16a

Ppp1r21

Ppp1r26Ppp1r3d

Ppp2ca

Ppp2r2b

Ppp2r2d

Ppp2r3a

Ppp2r3c

Ppp2r5d

Ppp3ca

Ppp3cb

Ppp3r1

Ppp4r1

Ppp4r1l−ps

Ppp6c

Ppt1

Pqlc2

Pqlc3

Pramef8

Prc1Prdm15

Prdm4 Prep

Prex1Prex2

Prickle1

Prim1

Prim2Prkaa1Prkab1

Prkab2

Prkacb

Prkar2bPrkcq

Prkd1 PrkdcPrmt10

Prmt2

Prnp

Procr

Prom2

Proser1

Prpf18

Prpf38a

Prpf38bPrpf39

Prpf40b

Prpf6

Prps1Prps2

Prr14l

Prr3

Prrg4Prss36

Prtn3

Prx

Psd4

Psen1Psen2

Psip1Psma8

Psmd13

Psme4Psrc1

PtafrPtar1Ptbp2Ptbp3

Ptcd2

Ptcd3

Ptdss1

PtenPtger3

Ptger4

Ptges3

Ptgr1Ptk2b Ptma

Ptp4a3

Ptplad1

Ptplad2

Ptpn12

Ptpn14

Ptpn22Ptpn23Ptpn4

Ptpn7

Ptprc

Ptpre

Ptprs

Ptprv Pum1Pum2

PuraPurbPus10

Pusl1

Pvr

Pvrl3

Pvrl4

Pvt1 Pwp1Pwp2

Pxk

Pydc4

Pygl

Pygm

Pygo1

Qars

Qk

Qpct

Qser1Qsox2

R3hcc1lR3hdm1RP23−175C13.3

RP23−175C13.6RP23−206J9.5RP23−378G4.2

RP23−441L19.1RP23−81C12.1RP24−465N15.1

Rab11a

Rab11�p1

Rab11�p3

Rab12Rab18Rab19

Rab20

Rab21 Rab27b

Rab2a

Rab2b

Rab31

Rab32

Rab33bRab34

Rab3gap1

Rab3il1Rab40b

Rab42

Rab43Rab5a

Rab8b

Rabep2

Rabepk

Rabif

Rabl3

Racgap1Rad1

Rad17

Rad21

Rad51Rad51ap1

Rad51d

Rad52

Rai14Rai2

Ralgapa1

Ralgapa2Ralgds

Ralgps2

Ramp1

Ramp2

Ran

Ranbp1

Ranbp2

Ranbp6 Rangap1

Rap1bRap1gap2

Rap1gds1

Rap2c

Rapgef2

Rarb

Rasa1

Rasa2

Rasa3Rasal3

Rasd1

Rasd2Rasgef1b

Rasgrp3

Rasip1

Rasl12Rassf2

Rassf4Rassf5

Rassf6Rb1

Rb1cc1Rbbp5

Rbbp6

Rbbp9

Rbck1

Rbfox1

Rbl1

Rbm10

Rbm11Rbm12b2

Rbm18

Rbm25 Rbm26

Rbm28

Rbm34

Rbm48Rbm4b

Rbms3RbpmsRbpms2

Rcan2

Rcan3

Rcbtb1

Rcc1

Rchy1

Rcn1

Rcn2Rcor3

Rdh10

Rdh11Rdh12

Rdh14

Rdm1Rdx

Rec8

Reck

Recql4Reep3

Reep4

Reep6Relb

Rell1

Relt

RenbpRetsat

Rev1

Rfc2

Rfc3

Rfc4 Rfc5

Rftn1

Rfwd3

Rfx2

Rfx3

Rfx7

Rgl1

Rgmb

Rgs1

Rgs18

Rgs19Rgs2Rgs7bp

RhagRhbdd1

Rhbdd3Rhbdf2

Rhbg

Rhebl1Rhob

Rhobtb1Rhobtb3

Rhof

RhojRhoqRhovRibc1Rictor Rif1

Rin2

Rinl Riok3

Ripk2

Rlf

RlimRmdn2

Rmnd5a

Rmnd5b

Rnase4

Rnase6

RnaselRnf103

Rnf11

Rnf114

Rnf115

Rnf121Rnf125

Rnf128Rnf13Rnf138

Rnf139

Rnf144a

Rnf168Rnf170

Rnf185Rnf19a

Rnf2

Rnf20

Rnf213

Rnf219Rnf34

Rnf41

Rnf6

Rnft1

Rngtt

Rnmtl1Rnpepl1

Robo4 Rock1

Rock2

Rora Rp2h

Rpa2

Rpf2RpgrRpl22l1

Rpn1

Rpp14

Rpp25Rpp40

Rps4y2

Rps6ka3

Rps6ka4Rps6ka5Rps6kl1

Rpusd1

Rpusd2

Rqcd1

Rrad

Rras2

Rreb1 Rrm1

Rrm2Rrnad1

Rrp1b

Rsad2Rsf1

Rsl1

Rsph9

Rspry1

Rsrc1Rtfdc1Rtn3

Rtn4

Rtp4Rufy1

Rufy4

Runx1t1

Runx2

Rwdd3

Rwdd4a

S100a4

S100a6

S100a8S1pr1S1pr2

Sacm1l

Sacs

Safb

Samd10

Samd8

Samd9l

Samhd1

Samsn1Sap130

Sar1b

Sars2

Sat1

Satb1

Sbf1

Sbspon

Sc4mol

Sc5dScaf11

ScaiScamp1

Scaper

Scara5

Scarb1

Sccpdh

Scd2Scfd2Schip1

Scml2Scnn1a

Sco1Scoc

Scrn3

Scyl3

Sdc1

Sdc2

Sdf2l1

Sdhaf2

Sdpr

Sdr42e1

Sdsl

Sec14l1Sec22a

Sec22c

Sec23b

Sec24d

Sec31b

Sec63

Secisbp2l

Seh1lSel1l

Selenbp1

Sell

Selp

Selplg

Sema3eSema6cSema6d

Sema7a

Senp5

Senp8

Sephs2

Sepn1

Sepp1

Sepsecs

Sept11

Sept2

Sept7

Serinc1

Serp1

Serpina3f

Serpina3g

Serpinb1a

Serpinb6aSerpinb6b

Serpinb8

Serpinb9

Serpine2Serping1

Sertad1

Sertad2Sesn1

Sestd1

Setd2Setd5

Setd7

Setmar

SetxSfpi1

Sfrp1

Sfrs18

Sgcb

Sgk1

Sgk3

Sgms1Sgol1Sgpl1

Sgpp1

Sgsh

Sgsm1

Sgsm2

Sgsm3

Sh2b1

Sh2d3c

Sh2d4aSh2d5Sh2d6

Sh3bgrl

Sh3bp5

Sh3d19

Sh3d21

Sh3gl2Sh3gl3

Sh3glb1

Sh3tc1

Sh3yl1

Shc4

Shcbp1Shcbp1l

Shisa5Shisa7

Shisa9

Shmt1

Shq1

Shroom1Shroom4

Siae

Sidt2Sigirr

Sik2

Sike1Sin3a

Sirt1

Ska1

Skil

Skint3Skiv2lSkiv2l2

Skp2

Sla

Slain1

Slamf1

Slamf8

Slc10a7 Slc11a2

Slc12a2

Slc12a6

Slc12a7Slc14a1

Slc14a2

Slc15a2

Slc15a4

Slc16a1

Slc16a12

Slc16a13Slc16a7

Slc17a8

Slc17a9

Slc18a2

Slc19a1

Slc1a4

Slc22a18

Slc22a3

Slc22a5Slc23a2

Slc25a13

Slc25a14

Slc25a15Slc25a16

Slc25a24

Slc25a25

Slc25a28

Slc25a32Slc25a36Slc25a44

Slc25a45

Slc25a46

Slc25a5

Slc25a53 Slc26a2

Slc27a1

Slc2a1

Slc2a12

Slc2a3

Slc2a8

Slc30a1Slc30a4

Slc30a5

Slc30a6Slc30a7

Slc31a1

Slc31a2 Slc35a1Slc35a3

Slc35b1

Slc35d1

Slc35d2

Slc35e1Slc35f5

Slc35f6

Slc37a3Slc37a4

Slc38a1

Slc38a9

Slc39a10

Slc39a11

Slc39a8

Slc39a9

Slc41a1

Slc41a3

Slc43a1

Slc43a2

Slc43a3

Slc46a1Slc46a3

Slc4a7

Slc52a2Slc52a3 Slc5a3

Slc5a6

Slc6a15

Slc7a4

Slc7a6

Slc8a1

Slc9a6

Slc9a8 Slco4a1

Slfn10−ps

Slfn2

Slfn5Slfn8

Slfn9

Slmo1

Smad1

Smad2Smad3

Smad4Smap2

Smarca1

Smarca5Smarcal1

Smc2

Smc4

Smc5Smc6

Smek1

Smek2Smg8Smim13

Smim15

Smim3

Smim5

Smo

Smox

Smpd2

Smpdl3a

Smtnl1

Smug1Smurf1

Smurf2Snap23

Snapc1Snapc3

Snapin

SncaSned1

Snora24

Snrnp200

Snta1Sntg2

Snx13

Snx17

Snx31Snx32

Snx4

Soat2

Socs2

Socs3Socs4

Socs6

Sorbs2

Sord

Sort1

Sos1Sos2

Sox18

Sox5

Sox6

Sox7

Sox8

Sp1Sp100 Spag5

Spag9

Spast

Spata1

Spata13

Spata2lSpata6

Spata7

Spats2l

Spc24Spc25 Spcs3Specc1 Specc1l

Speg

Spic

Spice1

Spin1

Spint1Spire2Spns1

Spo11

SpoplSpp1Sppl2a

Sppl2b

Spred1

Spry1

Spryd7Sptan1

Sptssa

Spty2d1

Sqle

Sqrdl

Sqstm1 Srd5a3

Srgap2

Srgap3

Srgn

Srm

Srms

Srp72

Srrm1

SrrtSrsf1

Srsf11

Srsf12Srsf2

Srsf6

Srsf7

Srxn1 Ssfa2

Ssh2

Ssh3

Ssr1

Ssr3

St14St3gal1St3gal2St3gal6

St6galnac4St7

St7l

St8sia4

Stag1Stam

Stam2 StambpStambpl1

Stap1

Stard10

Stard13Stard4

Stat1

Stat3

Stau2

Steap3

Stil

Stip1

Stk17b

Stk25

Stk3

Stk35

Stk36

Stmn1

StomStox2

Stpg2 StradaStradb

Strn3

Stt3aStx11

Stx12

Stx16Stx2Stx3 Stx7

Stxbp3aStxbp4

Stxbp5Stxbp6

Sucla2Suco

Sult1a1

Sult2b1Sumf1Sun1Supt7lSurf1Suv420h1

Suv420h2

Svip

Swap70

Swsap1

Swt1

Syk

Syncrip

Syne1

Syne2

Syne3

Sypl

Sytl4

Szt2

Tab1

Tab2

Tab3 Tacc3

Tada1

Taf1

Taf13

Taf1dTaf2

Taf7

Taf9b

Tanc2

Tango2Taok1

Taok2

Tap1

Tardbp

Tars

Tas1r1 Tasp1Tatdn2 Tbc1d1Tbc1d10c

Tbc1d14Tbc1d15

Tbc1d19Tbc1d22a

Tbc1d24

Tbc1d25Tbc1d5

Tbc1d7

Tbc1d8bTbc1d9

Tbccd1

Tbcd

Tbce

Tbcel

Tbck

Tbl1xr1

Tbxa2r

Tbxas1Tc2n

TcaimTceal8

Tceanc

Tcerg1

Tcf12Tcirg1Tcn2

Tcp11l2

Tctn2

Tdp2Tdrd7Tecpr1

Tecpr2

Tefm

Tek

Telo2Tenc1

Terf2ip TesTet1

Tex10Tex15

Tex30

Tex9

Tfcp2l1

Tfec

TfgTfpi

Tfr2

Tfrc

TgfbiTgfbr1

Tgfbr2

Tgm1

Tgoln1Tgtp1

Tgtp2

Tha1

Thbs1

Them4

Themis2

Thnsl1Thnsl2 Thoc1

Thoc2

Thoc5

Thoc6

Thop1

Thsd1Thumpd1

Tia1

Tial1

Ticam1

Tigd3

Tigd5

Timm44Timp2

Timp3Tinagl1

Tiparp

Tipin

Tiprl

Tirap

Tjp1Tk1Tk2

Tlcd2Tldc1

Tlr12Tlr2

Tlr4

Tm2d1

Tm7sf3Tm9sf2 Tm9sf3

Tm9sf4Tmc6Tmc8

Tmco1

Tmco3

Tmco4

Tmed5

Tmed7

Tmed8Tmem100

Tmem104

Tmem117Tmem125

Tmem129

Tmem132bTmem135

Tmem136Tmem14a

Tmem154

Tmem161a

Tmem161b

Tmem164Tmem165

Tmem167

Tmem167b

Tmem168

Tmem173

Tmem176aTmem177

Tmem18

Tmem180

Tmem184bTmem184cTmem185b

Tmem186Tmem194Tmem199

Tmem204

Tmem206

Tmem209

Tmem214

Tmem218

Tmem220

Tmem231

Tmem241Tmem245

Tmem26

Tmem260

Tmem29Tmem30a

Tmem33

Tmem38a

Tmem48

Tmem50b

Tmem55aTmem62

Tmem68

Tmem70

Tmem71 Tmem74bTmem79

Tmem86a

Tmem87b

Tmem97Tmf1

TmieTmlhe

Tmod2 Tmpo

Tmtc2

Tmtc3

Tmtc4

Tmub2 Tmx1

Tmx3

Tmx4

Tnfaip1Tnfaip2

Tnfaip3Tnfaip8Tnfaip8l1

Tnfrsf13bTnfrsf13c

Tnfrsf21

Tnfrsf25Tnfrsf26

Tnfsf10Tnfsf11

Tnfsf4Tnfsf9Tnik

Tnip3

Tnks2Tnni3

Tnpo1

Tnpo2

Tob1

TollipTop1

Top2a

Top2b

Top3b

Tor1aTor1b

Tor3a

Tox

Tpd52

Tpgs2

Tpm4

Tpp1

Tpp2Tpst1

Tpst2

Tpx2

Tra2b

Traf1 Traf2

Traf3

Traf3ip3

Trafd1

Traip

Trak2Tram1Tram1l1

Trappc11

Trappc13Trappc6b

Trbv12−2

Trdmt1

Treml1

Trib2

Trib3

Trim11

Trim12c

Trim13

Trim21

Trim23

Trim24Trim27

Trim30a

Trim30bTrim30d

Trim35

Trim37

Trim41Trim44

Trim45Trim46Trim47

Trim5

Trim59Trim68

Triobp

Trip11

Trip12Trip13

Triqk

Trit1

Trmt10c

Trmt11 Trmt12Trmt2b

Trnau1ap

Tro Troap

Trove2Trp53cor1

Trp53i11

Trp53inp1Trpa1

Trpc2

Trpc4ap

Trpc6

Trpm7

Trps1

Trpv2Trpv4

Trub2Tsc2

Tsc22d1

Tsc22d2Tsc22d3

Tsen2Tsga10

Tspan14

Tspan17

Tspan2Tspan6Tspan7

Tspyl1

Tspyl3

Tssc1

TstTtc12 Ttc14Ttc17

Ttc19

Ttc3

Ttc30a1

Ttc30a2

Ttc30b

Ttc33

Ttc38

Ttc39a

Ttc39b

Ttc4

Ttc5Ttc8

Ttc9c

Ttf2Tti1Ttll1Ttll10Ttll13 Ttll7

Ttpa

Tuba1b

Tuba4a

Tuba8

Tubb4b

Tubb5

Tubb6

Tubd1

Tube1

Tubg1Tubgcp2

Tuft1Tug1Tulp1

Tvp23b

Twsg1

TxlngTxndc16

Txndc5

Txnrd2

Tymp

Tyms

U2surp

Uap1

Uap1l1

Uba3

Uba5

Uba7

Ubac1

Ubap1

Ube2a

Ube2bUbe2c

Ube2d3

Ube2e2

Ube2e3Ube2g1

Ube2j1

Ube2l6Ube2q2

Ube2w

Ube3a

Ube4a

Ubiad1Ubl3 Ublcp1

Ubn1

Ubox5

Ubr1

Ubr7

Ubtd1Ubxn2a

Ubxn2b

Ubxn7 Uchl5

Uck1

Ufsp2

Ugdh

Uggt2

Ugp2

Ugt8a

Uhrf1Uhrf1bp1

Uhrf2

Ulk2

Unc45a

UngUnk

Upf3b

Upk1b

Upp1

Uqcc

Urb2Uri1

Usb1

Usp1

Usp11

Usp14

Usp18

Usp20Usp24

Usp25Usp27xUsp3

Usp31

Usp32

Usp37

Usp40

Usp46Usp49

Usp6nl

Ust

Utp15

Utp18

Utp23

Utp6

Uvssa

Vamp1

Vamp3

Vamp4

Vamp7

Vangl2

Vbp1

Vcpip1

Vdr

VegfcVezf1

Vhl

Vill

Vim

Vldlr

Vma21

Vmac

Vmp1

Vnn1

Vps11 Vps13a

Vps13b

Vps13d

Vps18

Vps26a

Vps29

Vps33b

Vps35

Vps37a

Vps37c

Vps39

Vps45

Vps54

Vstm5

Vwa5a

Vwf

Wars

Wars2Wasl

Wbp1

Wbscr16

Wbscr27

Wdfy1

Wdfy3

Wdpcp

Wdr13Wdr17

Wdr20a

Wdr25

Wdr34

Wdr36

Wdr41

Wdr43

Wdr44

Wdr45

Wdr45b

Wdr47Wdr5

Wdr59

Wdr62Wdr65Wdr7

Wdr78

Wdr81Wdr85

Wdr90

Wfdc2

Wipf1

Wls

Wrap53

Wrb

Wrn

Wsb1

Wsb2

WtipWwox

Wwp1

Wwp2Xab2

Xbp1

Xdh

Xiap

Xist

Xk

Xkr6

Xkr8

XlrXlr3a

Xlr3b

Xlr4aXlr4bXpc

Xpnpep1Xpnpep3

Xpo1

Xpr1Xrcc3

Xrn1Xrn2

Xxylt1

Xylt2

Yaf2

Yars

Yars2

Ybey

Ydjc

Yes1

Yipf5

Yipf6

Ylpm1

Yme1l1Yod1

Ypel5

Ythdf3

Yy1Zadh2Zbed6

Zbp1

Zbtb10

Zbtb16

Zbtb20

Zbtb21

Zbtb25Zbtb26

Zbtb33 Zbtb41Zbtb43

Zbtb44

Zbtb46Zbtb48

Zbtb6Zc2hc1a

Zc3h12bZc3h12c

Zc3h13

Zc3h14

Zc3h7a

Zc3hav1

Zc3hav1lZc4h2

Zcchc10

Zcchc11

Zcchc18Zcchc2

Zcchc24

Zcchc4

Zcchc6

Zcchc8

Zdhhc17

Zdhhc2

Zdhhc20

Zdhhc24Zdhhc6

Zeb2

Zfand1Zfand4

Zfand5

Zfand6

Zfc3h1

Zfp101

Zfp108Zfp11

Zfp111Zfp113

Zfp119aZfp119b

Zfp120

Zfp125Zfp141

Zfp142

Zfp143

Zfp148

Zfp157 Zfp160

Zfp180

Zfp182Zfp189

Zfp191

Zfp2Zfp202

Zfp207Zfp229

Zfp239

Zfp248

Zfp26

Zfp260

Zfp266Zfp273

Zfp275

Zfp277

Zfp28

Zfp280c

Zfp280dZfp287 Zfp296

Zfp3

Zfp30Zfp300Zfp318

Zfp324

Zfp329

Zfp341 Zfp35

Zfp354a

Zfp354c

Zfp36 Zfp367Zfp37

Zfp386

Zfp39

Zfp397

Zfp398

Zfp40

Zfp407Zfp41

Zfp420

Zfp426

Zfp429

Zfp442

Zfp449Zfp451

Zfp454Zfp455

Zfp456

Zfp458Zfp46

Zfp472

Zfp473

Zfp493

Zfp507Zfp51Zfp518aZfp518b

Zfp52

Zfp521

Zfp523

Zfp53

Zfp54Zfp551

Zfp558

Zfp563

Zfp568

Zfp574

Zfp583

Zfp592

Zfp595

Zfp597Zfp599

Zfp60

Zfp605

Zfp606

Zfp61Zfp612 Zfp617

Zfp619

Zfp62

Zfp623

Zfp637Zfp651

Zfp654

Zfp655

Zfp658Zfp661

Zfp667Zfp672

Zfp677

Zfp68Zfp704

Zfp708

Zfp709

Zfp71−rs1

Zfp711

Zfp712

Zfp715Zfp719

Zfp72

Zfp738Zfp746

Zfp747

Zfp748

Zfp758

Zfp759

Zfp760

Zfp763

Zfp764Zfp772

Zfp773Zfp775Zfp78Zfp783

Zfp784

Zfp788

Zfp799 Zfp800

Zfp808

Zfp81

Zfp820

Zfp825

Zfp830Zfp839

Zfp846Zfp85−rs1

Zfp866Zfp867

Zfp868

Zfp87

Zfp870

Zfp871

Zfp873

Zfp874a

Zfp874bZfp882Zfp91

Zfp92

Zfp93

Zfp930

Zfp931Zfp932

Zfp933

Zfp934

Zfp935

Zfp937Zfp938

Zfp939

Zfp940

Zfp941

Zfp942

Zfp943

Zfp944

Zfp945

Zfp946

Zfp947Zfp948

Zfp951

Zfp952

Zfp953

Zfp954

Zfp955b

Zfp958

Zfp959

Zfp961

Zfp97

Zfr

Zfyve1

Zfyve19

Zfyve27Zik1

Zkscan14

Zkscan4Zkscan8Zmat1

Zmpste24Zmym4

Zmym5

Znf41−ps Znrf2

Zranb2

Zscan12

Zscan18Zscan21Zscan22Zscan29

Zswim3

Zswim4Zswim5

ZufspZw10

ZwintZxdc

Zzef1

mmu−let−7d

mmu−mir−29b−2

mt−Nd1mt−Nd2mt−Rnr2

Component 1

Com

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Wilson et al Figure S3

Itga2

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-5 100 5 15

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10080604020

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Frac

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[%]

-5 100 5 15 -5 100 5 15 -5 100 5 15 -5 100 5 15

100806040200

100806040200

100806040200

Ct values (qPCR)

Tet2 Vwf

Procr Runx1 Spi1 Sh2b3 Smarcc1 Tal1 Tcf7

Mpl Myb Nfe2 Nkx2-3 Notch1 Pbx1 Prdm16

Kit Ldb1 Lmo2 Lyl1 Mecom Meis1 Mitf

G�1b Hhex Hoxa5 Hoxa9 Hoxb4 Ikzf1 Itga2b

Ets2 Etv6 Fli1 Gata1 Gata2 Gata3 G�1

Bptf Cbfa2t3h Cdkn2a Csf1r Dnmt3a Erg Ets1100

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0

10080604020

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Frac

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[%]

-5 100 5 15 -5 100 5 15 -5 100 5 15 -5 100 5 15Quantile normalized read counts (scRNA-Seq)

100806040200

100806040200

100806040200

-5 100 5 15

Tet2 Vwf

Procr Runx1 Spi1 Sh2b3 Smarcc1 Tal1 Tcf7

Mpl Myb Nfe2 Nkx2-3 Notch1 Pbx1 Prdm16

Kit Ldb1 Lmo2 Lyl1 Mecom Meis1 Mitf

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0.8

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CD48

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SLAM Scahi

CD34 c-Kit FLT3 EPCR

SLAM Scalo

SLAM Scahi

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Wilson et al Figure S4

−200 −100 0 100 200−200

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100

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LowHighSca-1 expression

0 200 400 6000

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Wilson et al Figure S5

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Supplemental Information

Figure S1, related to Figure 1; Functional validation of sorted populations for gene

expression profiling.

A) Sorting and gating strategies for the isolation of all HSC populations. B) Individual

progenitor cells were isolated and seeded in a methylcellulose-based colony assay. For CMP,

GMP and MEPs, 500 cells were isolated and placed into methylcellulose cultures. Colonies

were morphologically scored as mixed, erythroid, or granulocytic/monocytic and the

percentage of the correct colony type (e.g., granulocyte colony for GMP) is depicted. For

LMPPs, single cells were sorted onto OP9 feeder cells and scored for the presence of myeloid

and lymphoid elements. The percentage purity is indicated on the y axis. Error bars represent

data ± SEM. C) Each individual dot represents the donor chimerism at 16 weeks in a

recipient of 10 phenotypically identified HSCs. The phenotypes are indicated on the x-axis

and the % donor contribution on the y-axis. D) The stacked bar graphs depict the relative

contribution of the donor clone to each of the granulocyte/monocyte (GM, red), B-cell (B,

blue), and T-cell (T, green) lineages.

Figure S2, related to Figure 2; Robustness analysis.

t-SNE plot of all cells calculated from the 43 genes analysed by Fluidigm. Axes are in

arbitrary units. A) Individual populations are shown for clarity. B-C) t-SNE plot with the

MolO HSCs identified by the computational weighting highlighted in red. B) Leeway factor

of 70%. C) Leeway factor of 90%. D) Difference in normalised marker expression between

MolO cells and NoMO cells for each sorting strategy. Error bars correspond to 1 standard

deviation. First column represents all MolO cells compared to all other HSCs across sorting

strategies. * = p<0.05, ** = p<0.01. E) FACs plots for the individual HSC sorting strategies,

showing c-kit, Sca-1, CD48 and CD150. MolO HSCs are highlighted in red, NoMO HSCs

are highlighted in gray.

Figure S3, related to Figure 3; RNA-Seq analysis

A) Principal component loading plot, indicating the 4533 differentially regulated genes

identified from the scRNA-Seq. The 92 single cells which were analysed by scRNA-Seq are

shown within the plot as red circles. B) Importance of the individual genes for predicting

MolO cells using random forests, quantified as the total decrease in node impurity averaged

over all trees in the random forest (gini importance). C) ROC curve quantifying classification

performance and generalizability of MolO predictor using 10-fold cross-validation. An area

under the cross-validated ROC curve of 0.881 indicates that in 88% percent of predictions, a

randomly drawn MolO cell would receive a higher MolO score than a randomly drawn

NoMO cell. D) Distribution plots are shown for both the qPCR and quantile normalized

RNA-Seq read counts. Only genes assayed by qPCR are shown. Heterogeneous genes are

shown in blue. i) qPCR. ii) Normalised RNA-Seq read counts.

Figure S4, related to Figure 4; SLAM Scalo and SLAM Scahi populations predominantly

express other HSC markers.

A) Sorting strategy used to isolate SLAM Scalo and SLAM Scahi cells. CD48-CD150+ were

selected (left hand panel) and the cells staining positively for Sca-1 were divided into low and

high expression (right hand panel). FACs plot as in Figure 4a, shown here for reference. B)

Histograms showing the MFI for each additional HSC marker (CD34 (negative), c-Kit

(positive), FLT3 (negative), and EPCR (positive)) are shown for both SLAM Scalo (upper,

blue) and SLAM Scahi (lower, blue) cells. The blue populations are overlaid onto the red, the

latter showing the cell distribution of each marker for the whole bone marrow. Notably,

both SLAM Scalo and SLAM Scahi express all HSC markers as expected.

Figure S5, related to Figure 5; Linking molecular states with functional outcomes.

A) Sca-1 expression overlaid over the subset of sequenced cells shown in Figure 5d. The cells

in the lower left-hand cluster have significantly higher Sca-1 expression and significantly

higher MolO score compared to the cells in the upper right-hand cluster. Axes are in arbitrary

units. B) Joint t-SNE representation of RNA-Seq data and functional data coloured according

to SuMO score. Axes are in arbitrary units. C) t-SNE for 4 of the 5 patients, showing distinct

clusters of mammary luminal progenitor cells defined by hierarchical clustering in the 2D t-

SNE space; two of the bottom 3 clusters (5 and 6) are enriched for colony-forming cells. D) t-

SNE with overlay of luminal colony-forming. Red circles indicate successful colony

formation, blue circles did not form colonies. (Over-representation of colony presence in

clusters 5 and 6, p=0.00001). E) Bar graph showing the colony formation across all cells and

the post-enrichment colony formation (bottom clusters only) in individual patient samples

(A-D). A 5th sample was not enriched using the clustering tool. F) Differential expression

between enriched (5 and 6) and non-enriched clusters (1, 2, 3 and 4) of the t-SNE map for the

markers used to isolate the luminal progenitors.

Table S1, related to Figure 1; Fluidigm Taqman Assays.

A summary of the TaqMan assays used for single cell gene expression analysis.

Table S2, related to Figure 3; Differentially expressed genes as identified by scRNA-Seq.

The 4533 genes identified by scRNA-Seq to be differentially regulated between individual

single cells and for which the biological variability exceeded technical variability.

Table S3, related to Figure 3; Differentially expressed genes ranked based on their

MolO score.

Top 500 ranked scRNA-Seq genes by the MolO score. Top negatively correlated genes

(NoMO). Top positively correlated genes (MolO).

Table S4, related to Figure 5; Differentially expressed genes based on their SuMO score.

The top 500 ranked scRNA-Seq genes by the SuMO score. Top negatively correlated genes

(non-SuMO). Top positively correlated genes (SuMO).

 

Table S1: Fluidigm assaysGene name Assay ID

Bptf Mm01251151_m1Cbfa2t3h Mm00486780_m1Cdkn2a Mm00494449_m1 Csf1r Mm01266652_m1Dnmt3a Mm00432881_m1Egfl7 Mm00618004_m1Eif2b1 Mm01199614_m1Erg Mm01214246_m1Ets1 Mm01175819_m1Ets2 Mm00468977_m1Etv6 Mm01261325_m1Fli-1 Mm00484409_m1Gata1 Mm00484678_m1Gata2 Mm00492300_m1Gata3 Mm00484683_m1Gfi1 Mm00515855_m1Gfi1b Mm00492318_m1Hhex Mm00433954_m1Hoxa5 Mm00439362_m1Hoxa9 Mm00439364_m1Hoxb4 Mm00657964_m1Ikzf1 Mm01187882_m1Itga2b Mm00439768_m1kit Mm00445212_m1Ldb1 Mm00440156_m1Lmo2 Mm01281680_m1Lyl1 Mm01247198_m1Mecom Mm01289155_m1Meis1 Mm00487659_m1Mitf Mm01182480_m1Mpl Mm00440310_m1Myb Mm00501741_m1Nfe2 Mm00801891_m1Nkx2-3 Mm01199403_m1Notch1 Mm00435249_m1Pbx1 Mm04207617_m1Polr2a Mm00839493_m1Prdm16 Mm00712556_m1Procr Mm00440993_mHRunx1 Mm01213405_m1Spi1 Mm00488142_m1Sh2b3 Mm00493156_m1Smarcc1 Mm00486224_m1Tal1 Mm01187033_m1

Tcf7 Mm00493445_m1Tet2 Mm00524395_m1UBC Mm01201237_m1Vwf Mm00550376_m1

Supplemental Experimental Procedures

Purification of Stem and Progenitor Cells.

Suspensions of BM cells from the femurs, tibiae and iliac crest of 8–12-week-old C57BL/6

mice were isolated and depleted of red blood cells by an ammonium chloride lysis step

(STEMCELL Technologies, STEMCELL)). HSCs were isolated using the following

antibodies: CD45-FITC or APC-Cy7 (Clone 30-F11 Biolegend), EPCR-PE (Clone

RMEPCR1560, STEMCELL), CD150-Pacific Blue or PE-Cy7 (Clone TC15-12F12.2,

Biolegend), CD48-APC (Clone HM48-1, Biolegend), Sca-1-Pacific Blue or PE (Clone E13-

161.7, Biolegend), FLT3-PE or PE-Cy5 (Clone A2F10, eBioscience), CD34-FITC (Clone

RAM34, BD Biosciences), c-kit APC-Cy7 (Clone 2B8, Biolegend), and a panel of lineage

markers (Hematopoietic Progenitor Enrichment Cocktail, STEMCELL) plus streptavidin-

V500 (BD Biosciences). The side population (SP) cells were isolated according to the

detailed protocol found at

https://www.bcm.edu/research/labs/goodell/index.cfm?pmid=20017 and as previously

published (Challen et al., 2012). The Hoechst dye was obtained from Sigma and used at a

final concentration of 5 µg/ml. Cells were sorted using a BD Influx sorter equipped with 355

nm, 405 nm, 488 nm, 561 nm, and 640 nm lasers. For single cell gene expression assays,

cells were sorted into individual wells of 96 well PCR plates using a modified plate holder for

increased stability. For single cell transplantation and in vitro assays, cells were sorted into

individual wells of a U-bottom 96 well plate. For progenitor colony forming cell assays and

10-cell transplantation assays, cells were sorted into 1.5 ml tubes containing serum free

media.

Progenitor Cell Assays

500 CMPs (Lin-c-kit+Sca-1-CD34+FcγRlow), MEPs (Lin-c-kit+Sca-1-CD34-FcγRlow), and

GMPs (Lin-c-kit+Sca-1-CD34+FcγRhi) were isolated into 1.5ml Eppendorf tubes containing

serum-free medium. Cells were then divided into a high concentration fraction (~450 cells)

and a low concentration fraction (~45 cells) and placed into a semi-solid medium containing

growth factors to support growth of all myeloid colony types (MC3434, STEMCELL). Both

high and low concentration fractions were spread across 3 individual wells of a 6 well plate

and counted after 10 and 14 days of culture. Single LMPPs (Lin-c-kit+Sca-1+CD34+FLT3+)

were sorted into individual wells containing OP9 cells supplemented with 100 ng/ml IL-7 and

50ng/ml FLT-3. All wells were harvested at day 28 and analyzed for the presence of B-

(defined as B220+) and myeloid (Ly6g and/or Mac1) cells.

Single HSC Cultures

SLAM Scahi and SLAM Scalo HSCs were sorted and cultured in STEMSPAN medium

containing 300 ng/ml SCF, 20 ng/ml IL-11, Glutamine, Penicillin, Streptomycin, β-2-

mercaptoethanol and 10% FCS as described previously (Kent et al., 2008; Kent et al., 2013).

After 24 hours, wells were scored for the presence of a single cell and counted each day to

track the clonal growth of individual cells. For the immunophenotyping studies, clones were

individually stained and assessed for the expression of Sca-1, c-Kit, and a panel of lineage

markers along with 7-Aminoactinomycin D (7AAD, Invitrogen) to mark dead cells.

Clone Size Calculations and Antibody Information for In Vitro Cultures

When the clones began to appear, they were estimated to be small (50-5000 cells), medium

(5000-20,000 cells), or large (20,000 or more cells). No clones had fewer than 50 cells. 10-

day clones were stained with biotinylated lineage marker antibodies (Haematopoietic

Progenitor Enrichment Cocktail, STEMCELL), c-Kit-APC (BD) and Sca-1-Pacific Blue

(Biolegend). To enumerate cells, a defined number of fluorescent beads (Trucount Control

Beads, BD) were added to each well and each sample was back-calculated to the proportion

of the total that were run through the cytometer. Small clones were not able to be assessed

individually by flow cytometry and were pooled – the % of KSL cells was greater than 95%.

Flow cytometry was performed on an LSRII Fortessa (BD) and all data were analyzed using

Flowjo (Treestar, USA).

Single-Cell Gene Expression Analysis.

Single-cell gene expression analysis was performed as described previously (Moignard et al.,

2013). Briefly, single cells were sorted by FACS directly into individual wells of 96-well

plates containing lysis and preamplification mix. Reverse transcription and specific target

amplification were performed in the same plates 24 hours after sorting. cDNA was diluted

1:5 with TE before qPCR on the BioMark HD. For the qPCR, Taqman assays (Life

technologies) and cDNA samples were then loaded into a 48.48 Dynamic Array (Fluidigm),

and then transferred to the BioMark HD for qPCR.

Bioinformatic Analysis of Single-Cell Gene Expression Data.

Single-cell expression data were collected using the Fluidigm Data Collection software. ΔCt

values were calculated as previously described (Guo et al., 2010) by cell-wise normalization

to the mean expression level of two housekeeping genes (Ubc and Polr2a). Briefly, Ct values

were subtracted from the limit of detection of the BioMark (Ct 27) (Guo et al., 2010),

followed by subtraction of the mean Ct value of Ubc and Polr2a for each cell. The ΔCt value

for genes that were not expressed was set to be 3.5 cycles more than the lowest Ct value per

gene. All housekeepers, Cdkn2a and Egfl7 were removed from the dataset for downstream

analysis. Cdkn2a was not expressed in any of the cell types and Egfl7 assay experienced

technical issues. Hierarchical clustering was performed in R (www.r-project.org) using the

hclust package and heatmap.2 from the gplots package using Spearman rank correlations and

ward linkage.

t-SNE was performed in Matlab (Mathworks Inc., Natick, USA) using the Matlab

implementation (http://homepage.tudelft.nl/19j49/t-SNE.html) with standard settings.

We identified MolO cells based on a weighting matrix defined by repopulation probabilities

and the 2D t-SNE representation of the data. As cells with similar gene expression patterns

are located in close proximity in the 2D t-SNE representation of the data, we aimed to

identify a contiguous region in the t-SNE plot where cells within the region locally fulfil the

mixture constraint derived from the weighting matrix. We therefore iteratively assessed the

local neighbourhoods of all cells for molecular overlap and tested whether within each

neighbourhood no progenitors were present and the fraction of each HSC subtype exceeded

80% of its respective weight. We introduced the leeway factor of 80% to allow for some

sampling variation. Robustness analysis was performed additionally which assessed the

leeway factors of 70 and 90 percent (Figure S2b and S2c). If both conditions were met, the

cell was classified as MolO core cell and all cells within its neighbourhood were classified as

MolO cells. We performed a grid search to determine the neighbourhood size maximising the

number of density-reachable MolO core points (Ester, 1996).

Random forests were trained on the normalised Ct values of the set of genes which were

assayed by single-cell gene expression and variable above technical noise in scRNA-Seq.

Training was performed on all cells from sorting strategy HSC1 and generalizability was

quantified using 10-fold cross-validation (Figure S3c). For prediction of the sequenced cells,

read counts had to be transformed to the same scale as normalised Ct values. As both sets of

cells originated from the same sorting strategy we reasoned that the distributions of

expression values of variable genes should be similar for the qPCR and the RNA-Seq data

and performed quantile normalisation for the respective subsets of cells expressing each

variable gene (Figure S3d). Training and testing of the classifier was performed in python 2.7

using the sklearn library.

Single-Cell RNA-Seq

Single cell RNA-Seq analysis was performed as described previously (Picelli et al., 2014).

Briefly, single cells were sorted by FACS directly into individual wells of a 96-well plate

containing lysis buffer. Reverse transcription and PCR amplification were performed in the

same plates. The resulting PCR products were purified, the quality of the cDNA library

verified and libraries were prepared using the Illumina Nextera XT DNA preparation kit.

Pooled libraries were then run on the Illumina Hi-Seq 2500. Files were demultiplexed and

reads aligned to the mouse genome (mm9) using STAR (Dobin et al., 2013). HTSeq (Anders,

2014) was run on all samples to assign mapped reads to genes, using Ensembl genes as a

reference. Several QC steps were implicated, such that a single cell was required to have

>500k unique counts mapping to annotated features, <10% of the counts mapped to

mitochondria DNA and >4000 genes detected. Mapped reads were normalised using size

factors as described in Brenencke et al (Brennecke et al., 2013). To estimate technical noise

we further followed Brenencke et al (Brennecke et al., 2013) and fitted the relation between

mean read counts and squared coefficient of variation using the ERCC spike-ins (Life

technologies) (Figure 3a i). Genes for which the squared coefficient of variation exceeded

technical noise were considered variable. To analyse the robustness of PC1, we sub-sampled

the data by repeating the PCA based on a random subset of 86 cells. We then quantified the

consistency of the PCA representations by projecting the 5 outlier cells onto the new

principal components, and calculated the correlation between the sub-sampled PC1 and PC1

from all cells. We repeated this procedure 500 times and the correlation coefficient was

always greater than 0.98. If we omit the 5 outlier cells from the original PCA, the correlation

coefficient was 0.983, giving good confidence in the clusters described.

Transplantation of Haematopoietic stem cells

10-cell transplantations were performed in CD45.1 lethally irradiated C57Bl/6 recipients

along with 250,000 helper cells from the spleen of CD45.1/.2 mouse. All single cell

transplantations were performed by standard intravenous tail vein injection of sublethally

irradiated Ly5-congenic adult W41/W41 mice as previously described (Dykstra et al., 2007).

Peripheral blood samples were collected from the tail vein of some mice at 4 weeks and all

mice at 8, 16, and 24 weeks after transplantation and red blood cells lysed using ammonium

chloride (Stemcell technologies, Stemcell). All samples were stained with the following

antibody panel: Ly6g-Pacific Blue (Clone 1A8, Biolegend), Mac1-FITC (Clone M1/70,

Biolegend), CD3e-PE (Clone 17A2, BD), B220-APC (Clone RA3-6B2, eBioscience),

CD45.1-Alexa700 (Clone A20, Biolegend) and CD45.2-APC-Cy7 (Clone 104, BD)). Donor

and recipient cells were distinguished by their expression of CD45.1 or CD45.2 and any

double positive CD45.1 and CD45.2 events were excluded from the analysis of donor

contributions to specific whole blood cell (WBC) subsets (leftover helper cells and/or

doublets). Transplanted cells from which at least 1% of the WBCs and were derived at 16

and/or 24 weeks after transplantation were considered to be repopulated with long-term

reconstituting cells. HSCs were further discriminated according to previously described high

(alpha or beta) or low (gamma or delta) ratios of their proportional contributions to the GM,

B- and T-cell subsets of the circulating WBCs assessed at 16 weeks after transplantation

(Dykstra et al., 2007). Flow cytometry was performed on an LSRII Fortessa (BD) and all data

were analyzed using Flowjo (Treestar, USA).

Isolation and assessment of mammary progenitors

All primary human material was derived from 5 reduction mammoplasties at Addenbrooke's

Hospital, Cambridge, UK, under full informed consent and in accordance with the National

Research Ethics Service, Cambridgeshire 2 Research Ethics Committee approval

(08/H0308/178) as part of the Adult Breast Stem Cell Study. All tissue donors had no

previous history of cancer and were premenopausal (ages 20 to 23). Mammary tissue was

dissociated to single cell suspensions as previously described (Eirew et al., 2010). Single cell

suspensions of human mammary cells were treated to detect the enzyme activity of aldehyde

dehydrogenase (ALDH) using the Aldefluor Kit (StemCell Technologies) as per the

manufacturer's instructions. The cells were incubated with the following primary antibodies:

CD49f-PE/Cy7 (clone: GoH3), epithelial cell adhesion molecule (EpCAM)-PE (clone: 9C4),

and CD45-APC-Cy7 (clone: HI30), CD31-APC-Cy7 (clone: WM-59) (collectively known as

Lin-APC-Cy7), as well as 4',6-diamidino-2-phenylindole (DAPI) for viability. Hank's

balanced salt solution supplemented with 2% FBS (Gibco) was used as the diluent for all

antibody incubation and washing steps. Cells were sorted using a BD Influx. Single-stained

control cells were used to perform compensation manually and gates were set in reference to

fluorescence minus-one-controls. The ALDH+ gate was set in reference to control

populations incubated with the ALDH inhibitor, DEAB in addition to Aldefluor. Luminal

progenitor populations were seeded single cell into 96 well plates with 1 × 104 irradiated

NIH-3T3 feeder cells. The cultures were maintained in Human EpiCult-B (StemCell

Technologies) supplemented with 5% FBS (StemCell Technologies) and 50 μg/ml

gentamicin and maintained for 10 to 12 days. Colony presence was scored under a

microscope.

Supplemental References

Anders, S., Pyl, P, T., and Huber, W. (2014). HTSeq - A Python framework to work with high-throughput sequencing data. bioRxiv. Brennecke, P., Anders, S., Kim, J.K., Kolodziejczyk, A.A., Zhang, X., Proserpio, V., Baying, B., Benes, V., Teichmann, S.A., Marioni, J.C., et al. (2013). Accounting for technical noise in single-cell RNA-seq experiments. Nat Methods 10, 1093-1095. Challen, G.A., Sun, D., Jeong, M., Luo, M., Jelinek, J., Berg, J.S., Bock, C., Vasanthakumar, A., Gu, H., Xi, Y., et al. (2012). Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 44, 23-31. Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21. Dykstra, B., Kent, D., Bowie, M., McCaffrey, L., Hamilton, M., Lyons, K., Lee, S.J., Brinkman, R., and Eaves, C. (2007). Long-term propagation of distinct hematopoietic differentiation programs in vivo. Cell Stem Cell 1, 218-229. Eirew, P., Stingl, J., and Eaves, C.J. (2010). Quantitation of human mammary epithelial stem cells with in vivo regenerative properties using a subrenal capsule xenotransplantation assay. Nat Protoc 5, 1945-1956. Ester, M., Kriegel, H, -P,. Sander, J, Xu, X. (1996). A density-based algorithm for discovering clusters in large spatial database with noise. Proceedings of Second International Conference on Knowledge Discovery and Data Mining, 226-231. Guo, G., Huss, M., Tong, G.Q., Wang, C., Li Sun, L., Clarke, N.D., and Robson, P. (2010). Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev Cell 18, 675-685. Kent, D.G., Dykstra, B.J., Cheyne, J., Ma, E., and Eaves, C.J. (2008). Steel factor coordinately regulates the molecular signature and biologic function of hematopoietic stem cells. Blood 112, 560-567. Kent, D.G., Li, J., Tanna, H., Fink, J., Kirschner, K., Pask, D.C., Silber, Y., Hamilton, T.L., Sneade, R., Simons, B.D., et al. (2013). Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion. PLoS Biol 11, e1001576. Moignard, V., Macaulay, I.C., Swiers, G., Buettner, F., Schutte, J., Calero-Nieto, F.J., Kinston, S., Joshi, A., Hannah, R., Theis, F.J., et al. (2013). Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis. Nat Cell Biol 15, 363-372. Picelli, S., Faridani, O.R., Bjorklund, A.K., Winberg, G., Sagasser, S., and Sandberg, R. (2014). Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181.