combined single-cell functional and gene expression analysis
TRANSCRIPT
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
pone
nt 2
Wilson et al Figure S3
Itga2
bVw
fPr
ocr
Ets2
Gat
a1M
ybPb
x1 Fli1
Bptf
Gfi1
bH
hex
Gat
a3Sp
i1D
mnt
3aLy
l1N
otch
Gfi1
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Impo
rtanc
e sc
ore
[au]
-5 100 5 15
10080604020
0
10080604020
0
10080604020
0
10080604020
0
Frac
tion
of c
ells
[%]
-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
80604020
0
10080604020
0
10080604020
0
10080604020
0
Frac
tion
of c
ells
[%]
-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
G�1b Hhex Hoxa5 Hoxa9 Hoxb4 Ikzf1 Itga2b
Ets2 Etv6 Fli1 Gata1 Gata2 Gata3 G�1
Bptf Cbfa2t3h Cdkn2a Csf1r Dnmt3a Erg Ets1
A B
C
D i) ii)
ROC curve Random Forest, AUC_CV=0.8811.0
0.8
0.6
0.4
0.2
01.00.80.60.40.20
TPR
FPR
CD150
CD48
FSC
SCA
SLAM Scalo
SLAM Scahi
CD34 c-Kit FLT3 EPCR
SLAM Scalo
SLAM Scahi
A
B
Wilson et al Figure S4
−200 −100 0 100 200−200
−150
−100
−50
0
50
100
150
200
LowHighSca-1 expression
0 200 400 6000
100
200
300
400
LowHighSuMO score
A B
Wilson et al Figure S5
C
Cluster 1
Cluster 2
Cluster 3
Cluster 4
Cluster 5Cluster 6
CloneNo clone
D
Marker expressionPatient sample
50
40
30
20
10
0A B C D
Pre-enrichmentPost-enrichment
Colo
ny fo
rmin
g effi
cien
cy (%
) FE
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.