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Per3. 0,0. 0,5. 1,0. 1,5. Bmal1KO. Bmal1WT. Bmal1KO. Bmal1WT relative mRNA expression. Ccnd2. Tgm3. H19. Ccna2 Ccnb1 Chek1 Rad9 Gadd45b Flg2. Lor.
SUPPLEMENTARY INFORMATION

doi:10.1038/nature10649

Supplementary Fig. 1 P19 Telogen

P22 Anagen onset

anti-GFP

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Supplementary Figure 1: Bulge stem cells are heterogenous in their clock. Immunohistochemistry of venus-GFP in Per1-venus mouse backskin at different hair cycle stages shows venusbright (arrow) and venusdim (arrowhead) cells in the bulge (Bu, bulge). Scale bar, 100 μm.

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RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 2 10 4

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Supplementary Figure 2: Per1-venus expression is homogeneous in the basal epidermis during anagen. a,b, Quantification of venusbright and venusdim cells in the α6 integrinbright/CD34- epidermal population of Per1-venus mice at different stages of the hair cycle (P19, P22, P25, P28 and P31) by FACS showed no changes in the percentage of venusbright and venusdim cells in the basal epidermis (mean ± s.e.m.; n ≥ 6).

2 | WWW. NATURE . COM / NATURE

SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 3 a

Telogen P19

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Supplementary Figure 3: Dynamic changes of Clock gene expression during the hair cycle in Per1-GFP reporter mice. a, Whole mount immunostaining of mouse tailskin from Per1-GFP mice at different stages of the hair cycle (from P19 to P31) showed a steady increase of GFPbright cells in the bulge compartment during anagen. Upper panel, anti-GFP staining; lower panel, direct GFP fluorescence. Scale bar, 100 μm. b, Immunostaining for GFP (red) and Keratin 14 (K14; green) revealed strong GFP expression in the isthmus region, which contains epSCs that feed into the epidermis and sebaceous glands. Scale bar, 50 μm. Bu, bulge; SG, sebaceous gland; Ist, isthmus; UI, upper isthmus; and HG, hair germ.

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RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 4 a a6-integrinbright/CD34+ LD

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Supplementary Figure 4: GFP/venus expression changes in a circadian manner. a, b, Comparison of venus mean fluorescence intensity in α6-integrinbright/CD34+ bulge (a) and α6-integrinbright /CD34- epidermal cells (b) from P27 Per1-venus mice kept under LD and DD conditions (one representative FACS profile is shown at CT6 and CT18 from each group; the gate to separate venusbright from venusdim cells was placed in all profiles according to the time point of brightest venus fluorescence intensity at CT12). The graph in b shows quantification of α6-integrinbright/CD34- epidermal cells at 4 different time points by FACS (mean ± s.e.m.; n = 2). White and black bars represent subjective day and night. c, Timelapse confocal imaging of telogen backskin of Per1-venus mice reveals an increase in venusbright nuclei in the bulge during the subjective night (from ZT13 to ZT24; arrows) Scale bar, 25 μm. Graph shows quantification of mean fluorescence intensity of venusbright nuclei in the bulge over 24 hours (n = 5 nuclei; mean ± s.e.m.; *** p < 0.001 using Cosinor analysis) d, Whole mount immunostaining of mouse tailskin from Per1-GFP mice at Zeitgeber time ZT3 (3 hours of light) or ZT12 (12 hours of light) shows clear differences in GFP intensity in the basal epidermis (arrows and higher magnification) comparing morning (ZT3) to the evening (ZT12). Scale bars 100 μm. CT, circadian time; ZT, Zeitgeber time; LD, 12 h light/12 h dark condition; DD, 12 h dark/12 h dark condition. 4 | WWW. NATURE . COM / NATURE

SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 5 a

Chromatin immunoprecipitation from tail epidermis 0,5

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Supplementary Figure 5: Bmal1 modulates the response of epidermal cells to activation and dormancy cues. a, ChIP from tail epidermis of Bmal1WT mice revealed binding of Bmal and Clock to E-box and E-box-like elements in the promoters of genes involved in adhesion, cell cycle, Notch, TGF-β and Wnt pathways. ChIP for Bmal1 and Clock in 2 regions of the circadian-regulated gene Dbp was used as positive (P, promoter) and negative control (Ex3, exon 3). (n = 3, mean ± s.e.m.). b, ChIP from FACS-purified bulge cells of Bmal1WT mice shows strong binding of Bmal to the promoters of genes highly expressed in the bulge SC compartment (n = 2; mean ± s.e.m.). Graphs in a and b show the percentage of immunoprecipitated DNA over the input. c, The differential expression of adhesion, Wnt and TGF-β pathway genes in primary keratinocytes isolated from newborn Bmal1WT (WT) and Bmal1KO mice (KO) was not statistically significant. Fold-change values are displayed as relative expression to Bmal1WT cells at 0h after normalization to Pumilio1 (Pum1) (n = 3). Results are shown as mean ± s.e.m., * p < 0.05 (two-way ANOVA with Bonferroni post-test). d, Real time PCR of adhesion, cell cycle, Wnt and TGF-β pathway genes in FACS purified bulge cells of 9-month-old Bmal1WT (WT) and Bmal1KO mice (KO) at ZT0 and ZT12. Expression levels are shown as relative mRNA levels after normalization to Pumilio1 (Pum1) (n = 2). Results are shown as mean ± s.e.m., * p < 0.05 (two-tailed Student’s t-test). w w w . n a t u r e . c o m / NATURE | 5

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 6 a

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Supplementary Figure 6: Conditional deletion of Bmal1 in the epidermis. a, Immunostaining for Keratin 14 (green) to mark Cre-expressing cells in the epidermis. Scale bar, 50 μm. b, Efficiency of LoxP recombination in the epidermis of K14Cre/Bmal1LoxP/LoxP (Bmal1KO) mice was assessed by PCR from genomic DNA of epidermal cells (left panel; lane 1, conditional allele; lane 2, Wt allele; lane 3, recombined allele) and by western blot for Bmal1 in keratinocytes isolated from Bmal1WT or Bmal1KO mice (right panel; arrow). c, Conditional deletion of Bmal1 effects the expression of venus in the epidermis of Bmal1KO/Per1-venus mice. Quantification of venusbright and venusdim cells in the α6 integrinbright/CD34+ bulge and α6 integrinbright/CD34epidermal population by FACS revealed a loss of venus expression in Bmal1KO/Per1-venus mice at ZT0 and ZT12 as compared to Bmal1WT/Per1-venus mice at ZT12 (numbers show percentages; n ≥ 3).

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SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 7

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Supplementary Figure 7: Primary mouse keratinocytes did not respond to activation through canonical Wnt signaling. Primary mouse keratinocytes of Bmal1WT and Bmal1KO mice transfected with TOP-Flash and FOP-Flash reporter plasmids were incubated for 24 hours in the absence or the presence of the canonical Wnt small molecular activator BIO (mean ± s.e.m.; n = 2).

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RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 8 a

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Supplementary Figure 8: Per1/2dKO epidermal cells show enhanced expression of Bmal1 targets. a, Backskin of Per1/2dKO mice and wildtype control animals was stained for Lef1 and Sox9 (n ≥ 6). Scale bars, 25 μm. b, Protein extracts from tail epidermis of 6-week-old Per1/2dKO mice and wildtype control animals (WT) were analysed by western blot for the expression of Lef1 and Sox9. Lanes represent extracts from two WT and two KO animals (#1, #2). Tubulin expression was analysed as loading control. c, Real time PCR of adhesion, Wnt and TGF-β pathway genes in primary keratinocytes of Per1/2dKO and wildtype control animals (WT). Fold-change values are displayed as relative expression (mean ± s.e.m.) to WT cells at 0h after normalization to Pumilio1 (Pum1) (n = 2). Results are shown as mean ± s.e.m., * p < 0.05, ** p < 0.01, *** p < 0.001 (two-way ANOVA with Bonferroni post-test).

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SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 9 a

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Supplementary Figure 9: Clock perturbation in vivo results in changes in the number of dormant bulge stem cells and premature tissue aging. a, Depilation-induced anagen entry shows a growth delay in Bmal1KO mice (n ≥ 5). b, Wholemount immunostaining for BrdU (LRCs) and the proliferation marker Ki67 of 10-week-old Bmal1WT and Bmal1KO after 1 week of TPA treatment (n = 5). c, Histological analysis of 5 month old Bmal1WT and Bmal1KO mice. Scale bars 100 μm. Results are shown as mean ± s.e.m., * p < 0.05, ** p < 0.01 (two-tailed Student’s t-test).

w w w . n a t u r e . c o m / NATURE | 9

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 10 a

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Supplementary Figure 10: Clockhigh epidermal keratinocytes have an increased clonogenic potential. a, b, Clonogenic assay of FACS-purified α6-integrinbright/ CD34- epidermal keratinocytes from the backskin of (a) Bmal1WT and Bmal1KO mice or (b) Per1/2dKO mice and wildtype control animals (1x105 cells seeded on J2 3T3 feeder cells).

1 0 | WWW. NATURE . COM / NATURE

SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 11

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Supplementary Figure 11: Percentage of bulge stem cells is not altered in Bmal1KO and Per1/2dKO mice. a, b, FACS analysis of α6-integrinbright/CD34+ bulge cells of (a) Bmal1WT and Bmal1KO mice or (b) Per1/2dKO mice and wildtype (WT) control animals show no differences in the percentage of bulge stem cells in 6-week-old animals (n = 4). Results are shown as mean ± s.e.m.; statistical significance was assessed by two-tailed Student’s t-test. NS, not significant.

w w w . n a t u r e . c o m / NATURE | 1 1

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 12 Bmal1WT

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Supplementary Figure 12: Bmal1KO and Per1/2dKO mice show opposite phenotypes with regard to epidermal proliferation. Whole mount immunostaining for the proliferation marker Ki67 shows lower expression of Ki67 in the epidermis of Bmal1KO mice compared to Bmal1WT mice, and enhanced expression in the epidermis of Per1/2dKO mice with regard to their wildtype controls. Scale bars, 100 μm. (Upper panel: unspecific staining in sebaceous glands was Ki67 antibody batch dependent).

1 2 | WWW. NATURE . COM / NATURE

SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 13 Bmal1WT

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Supplementary Figure 13: Analysis of aged Bmal1KO mice for the expression of p16, p19 and apoptosis. Immunohistochemistry for p16 in 10-12-month-old Bmal1WT and Bmal1KO mice revealed enhanced expression of p16 in the epidermis and hair follicles of Bmal1KO mice. Cells positively stained for p19 (black arrows) were only occasionally observed in Bmal1WT and Bmal1KO mice in similar numbers. Similarly, apoptotic cells (Tunnel; green; white arrows) were only infrequently detected and located mainly to terminal differentiated areas of the hair follicle and the interfollicular epidermis. Scale bars, 50 μm.

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RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 14 a

relative mRNA expression

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G-protein coupled receptor signaling

hemopoiesis

sensory perception/ cognition

fatty acid metabolic process

response to organic substance

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Supplementary Figure 14: Validation of microarray data comparing aged BMAL1WT and BMAL1KO mice. a, c, d, Validation of microarray data in FACS isolated α6 integrinbright/CD34- epidermal cells from Bmal1KO and Bmal1WT mice by real time PCR (n = 2-3, for each group) shows differential expression of (a) several core circadian genes, (c) genes implicated in cell cycle, DNA damage and terminal differentiation and (d) the abundance of the pri-miRNAs 23b, 27b and 24-1. Fold-change values are displayed as relative expression to Bmal1WT cells after normalization to Pumilio1 (Pum1), or 18S-rRNA. Results are shown as mean ± s.e.m., * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). b, Graphic depicts GO enriched biological processes of genes up- and down-regulated in Bmal1KO mice. (Fold change ≥1.4 or ≤-1.4; p-value ≤ 0.05).

1 4 | WWW. NATURE . COM / NATURE

SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 15 early stage

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Supplementary Figure 15: Loss of Bmal1 reduces the development of squamous tumors. a, Bmal1KO/K5-SOS mice developed fewer neoplastic lesions compared to Bmal1WT/K5-SOS littermates at early and late stages. b, Immunostaining of tumors of Bmal1WT/K5SOS and Bmal1KO/K5-SOS mice for terminal differentiation (Involucrin, Loricrin), apoptosis (Tunnel; arrows) and proliferation (Ki67). Scale bars, 100 μm.

w w w . n a t u r e . c o m / NATURE | 1 5

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 16 a

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β-catenin

Bmal1WT/K5-SOS

b

β-catenin

positive control (anagen HF)

Supplementary Figure 16: Absence of nuclear β-catenin in K5-SOS induced tumors. a, Immunostaining of sections from squamous tumors for β-catenin (red) shows no nuclear localization of β-catenin in K5-SOS induced tumors. b, Anagen hair follicle stained for nuclear β-catenin (arrow) was used as positive control to verify the staining protocol. DAPI (blue), scale bar, 75 μm.

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SUPPLEMENTARY INFORMATION RESEARCH

10 3

250K

200K

200K

150K

150K

FSC-H 100K

SSC-A 100K

Pacific Blue-A 10 2

50K

50K

10 5

10 5

4

10 4

10

CD34-APC-A

SSC-A

DAPI

10 4

250K

FSC-H

10 5

Epcam-APC-Cy7-A

Supplementary Fig. 17

3

10 APC-Cy7-A

10 2

10 2 0

0

0

50K

100K

150K

FSC-A

200K

250K

0

0

0 0

50K

100K

150K

FSC-A

200K

250K

0

50K

100K

150K

FSC-A

200K

250K

APC-A 10 3

0

10

2

10

3

10

4

lin neg. PE-A

10

5

0

10

2

10

3

10

4

10

5

α6-integrin-FITC-A

Supplementary Figure 17: Gating strategy for FACS analysis of K5-SOS induced tumors. Viable cells were selected by DAPI exclusion (gate 1), contaminating fibroblasts were excluded by forward and side scatter (gate 2), doublets were discarded in gate 3, epithelial cells were selected in gate 4 by a combination of a lineage negative markers in PE (CD31, CD45 and CD140a) and a positive marker (Epcam APC-Cy7), and α6bright/CD34+ tumorinitiating cells were selected in gate 5.

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RESEARCH SUPPLEMENTARY INFORMATION

Supplementary Table 2 Gene

Chromosome

Distance from transcriptional start site

Location

Motif

Validated by ChIP

TGF-β pathway Tgfb1

7

- 642

Promoter

CACGTG

no

Tgfb2

1

- 50/ + 440

Promoter/Exon1

CACGTG /AACGTG

no/ no

Smad7

18

- 4839/ -177

Promoter/ Promoter

CACGTG/ CACGTG

yes/ no

Smad9

3

- 2797/ + 262

Promoter/ Intron 1

CACCTG/ CACGCG

no/ no

Smurf2

11

- 4687/ - 3945

Promoter/ Promoter

CACGTG/ CACCTG

yes/ no

Lefty

1

- 297

Promoter

CACGTG

Dkk1

19

- 413

Promoter

AACGTG

no

Dkk3

7

- 874

Promoter

CACGTG

yes

Wnt3

11

- 2940/ - 400

Promoter/ Promoter

CACGTG/ CACGTG

no/ yes

Wnt10a

1

- 2533/ - 1410

Promoter/ Promoter

CACGTG/ CACGTG

no/ no

Lef1

3

- 4917/ - 4468/ - 4098

Promoter/ Promoter/ Promoter

CACGTG/ CACGTG/ CACGTG

yes/ no/ no

- 3243/ - 2501/ - 490

Promoter/ Promoter/ Promoter

CACGTG/ CACGTG/ CACGTG

no/ no/ no

yes

Wnt pathway

Ctnnb1

9

- 2596

Promoter

Dab2

15

- 2842/ - 1247

Promoter/ Promoter

AACGTG

Fzd2

11

- 361

Promoter

AACGTG

yes

Tcf4

18

- 2914

Promoter

CACGTG

yes

Sox9

11

- 44

Promoter

AACGTG

yes

Lhx2

2

- 4668/ -2591/ - 2040

Promoter/ Promoter/ Promoter

Notch2

3

- 2687

Promoter

Hes1

16

- 1809/ - 1014

Promoter/ Promoter

CACGTG/ CACGTG

- 4760/ - 3064

Promoter/ Promoter

AACGTG/ AACGTG

no/ no

- 1689/ + 150

Promoter/ Exon1

CACGTG/ AACGTG

no/ yes

- 251/ - 197/ -177

Promoter/ Promoter/ Promoter

- 90/ + 414

Promoter/ Intron 1

CACGTG/ AACGTG

no/ yes

- 656/ - 442

Promoter/ Promoter

CACGTG/ AACGTG

yes/ no

- 11/ + 186

Promoter/ Exon1

CACGTG/ CACGTG

no/ no

AACGTG/ AACGTG

CCCGTG/ CACGTG/ AACGTG

no no/ no

yes/ no/ no

Notch pathway CACGTG

yes no/ no

Adhesion/ Cell cycle/ others Itga6

Cdk4

Wee1

2

10

7

CACGTG/ CACGTG/ CACGTG

yes/ yes/ yes

Bmpr1a

14

- 4916

Promoter

AACGTG

no

Foxo3a

10

- 2435

Promoter

CACGTG

no

Gene promoters analyzed, but no binding sites for Bmal/ Clock found: Delta1, Filaggrin, Fzd3, Gli2, Hey1, Involucrin, Jagged1, Lgr5, Ltbp2, Ltbp3, Ltbp4, Nfatc1, Notch1, Sfrp1, Smurf1, Tcf3

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Supplementary Table 4 Real time PCR primer

 

Gene Forward CTGCCCGAACATCTGAAATTGA Bhlhe41 CTTTACCCGCAGCAAGAAAAC Ccna2 CTCAGGGTCACTAGGAACACG Ccnb1 CTGTGCGCTACCGACTTCAA Ccnd2 AAGGCTGGGTGAAGACCCTTA Cd34 CCCTGCCACCCTTACCAGA Cdkn2b (p15) GTTAAGCCACGAGAATGTAGTGA Chek1 AGGCCAAGCTAATCGGTATTGA Dab2 CCTGATCCCGCTGATCTCG Dbp CTCGGGGGTATTTTGCTGTGT Dkk3 GCCAACTGTCCAGTCTTGTTT Flg2 GTTCTTCTCGCAAGAGGAGAC Fzd2 CAACGCGGTTCAGAAGATGC Gadd45b AGGATGACAGGTGTGGTCAA H19 TGCAGAGGGCGAACAGAAC Itga6 TGTTTATCCCATCACGGGTGG Lef1 GATCGTCCCAACAGTATCAGTG Lor CTGATGTCCAACGCTTTGCC Ltbp2 ATCATTGACCGCTCCTTTAGGT Mki67 TACATTGGCTCTAGTGGCTCC Nr1d1 ACCAGGTCATTAAGTGTGTGC Per1 AAAAGCACCACGGATACTGGC Per3 AGGCGTTAGCATGGTGGAGTA Pum1 CACTGCAAGTATGGGGTCAAG Rad9 CCCCCACTGGATGACTACAG Smad3 GAAACCGGGGGAACGAATTAT Smad7 GAGCCGGATCTGAAGAGGGA Sox9 ACGAGCTGATCCCCTTCCA Tcf3 AGTGATGTCATGGCCAGCGAC Tgfbr2 TCAGTGCTCCCATCGGATTG Tgm3 GCTCAACGCCAACACAGTG Wnt10a CTCCTCTCGGATACCTCTTAGTG Wnt3a 48 miR primer (Sun et al., 2009) TTCTAAAGGAGGCTGCACTGC Pri-miR-23b GTTCTAAAGAGGGATTACCAC Pri-miR-27b CTAAAGGGTCCAGGTCTCCATG Pri-miR-24-1 CCTGGATACCGCAGCTAGGA 18S-RNA

Reverse GCTGCTCAGTTAAGGCTGTTAG ACGTTCACTGGCTTGTCTTCTA AGCTCTTCGCTGACTTTATTACC CACATCAGTGTGGGTGATCTTG TGAATGGCCGTTTCTGGAAGT CAGATACCTCGCAATGTCACG GATACTGGATATGGCCTTCCCT AATGTTGACCCAGATTCTTTGCT CAGGCACCTGGACTTTCCTT TCCTCCTGAGGGTAGTTGAGA GCCTTTCATTAGGGCTGAATCC TCGCTGCATGTCCACTAAATAG GGTCCACATTCATCAGTTTGGC TGAGTGAGTGGGTGGACAAT GCACACGTCACCACTTTGC CATGGAAGTGTCGCCTGACAG TGCTGAGAGGAGTAATAGCCC GTGGGGGTGAAGACGACTTT GCTCGCCTTGATGGTTCCT CAGTAGGTGATGGTGGGAAGTA CTCTCCCGGTCTTGCTTCA GGGAGGCTGTAGCTTGTCA TCCATCAAACGTACCCTTGTTC GCAATAAGTGAGGGCATGAGG TCCATCTTCACTCAGGTAGCC CGCGAGTCTTCTCCTCCCA GCTTGACGTGTGGCTTGTTC CAGGGACGACTTGACCTCAT CGCAGACTTCATGCGGCTTCTC GGCGTGGTTACTCATAAAGACAT CGAAAACCTCGGCTGAAGATG GCATGATCTCCACGTAGTTCCTG AAATCAGCATGCCAGGAACCCAAGC TGGTGAGCATCTTTGAAGGCTGTTG CTGAGCCAGTGTGTGAAATGAGAAC TCTAGCGGCGCAATACGAATG

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RESEARCH SUPPLEMENTARY INFORMATION

Supplementary Table 5 Chromatin immunoprecipitation primer

 

Gene Primer name Forward Dbp Dbp_P ACACCCGCATCCGGTAGC Dbp Dbp_E3 CGTGGAGGTGCTTAATGACCTTT 49 Dbp primer (Ripperger et al., 2006) Wnt3 Wnt3_UP CAACCCCTTTGCTTTCTAG Lef1 Lef1_farUP1 CAGTGTACTGTAACAGTG Dab2 Dab2_farUP CACTATGGAAAACGGGAC Dab2 Dab2_UP GAACCAGGACTGCACACT Notch2 Notch2_farUP CACTCCAACAATTCAACAG Itga6 Itga6_E1 GCTTCTAGCCCGGCTGGG Cdk4 Cdk4_UP1 CTTGTGCTCCACCCTCTC Cdk4 Cdk4_I1 GTGATCCTCTTTGTGCCTAG Lhx2 Lhx2-farUP1 CAGATCGCTGCAGGGAGAAG Tcf4 Tcf4_farUP CCAAGAAGTCTGCGGTGA Fzd2 Fzd2_farUP GCTCTTGTACCTCAGAGG Smad7 Smad7_farUP GGACATCAGCTCTGAATC Lefty Lefty_UP GCAGAGAACGTGAGACCTC Dkk3 Dkk3_UP TGCTGCAACGGACAGACC Sox9 Sox9_UP ATCCGGTCCAATCAGCGAC

 

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Reverse CCACTTCGGGCCAATGAG CATGGCCTGGAATGCTTGA GACACCCCCTTCCCAGTC CTTCTTCCAGCCTGCTTC CTCTTTGTTATTACACTTC GGAGGACACAAAAGCATC ACCCAACTTTAACTTCTG GAACTCACAGCCGCTTGTC CTGACGGCCACGTGACCTG GCCAACGCGATCAGCAAC GGTCATACCATACAGAGAT CCAGCGATCATTAGTTACC GCTCAGTCCGTGATTGCT AGGCTGGTATGATGATGG TGTTAGCCATATACTTCACC CTCTCCGCATCTCCTGATC CTCTCCGACTTCCAGCTCAG