Supplemental Figure 1 A B

30 20 plaftorm microarray RNA−seq 25 scRNA−seq 10 Maryanovich 20 Mann Sun Lazare Kirshner Flach 0 Norddahl Bersenev Grover Wahlestedt Kowalczyk espan (months)

10 PC2 − 20.19% li f −10

5 Chambers 1 −20 young aged −20 −10 0 10 20 PC1 − 47.95%

C fold-chage - ReAnl D h-Chambers Wahlestedt 7.5 10 6

5.0 5 4

0 2.5 2 logFC FC−ReAn

−5 2 0.0 0 log −10 −2.5 −2

v 2 2

er −5.0 R = 0.82188 R = 0.88604 v vich Sun Flach Mann −5.0 −2.5 0.0 2.5 5.0 7.5 −2 0 2 4 6 Gr o Lazare

an o Sun Grover Kirshner y ow alczyk Norddahl ahlestedt Bersen e Chambers K 2 W 6 6 R = 0.44023 Ma r

4

4 2

FC−ReAn 0 2 2 log −2

−4 R2 = 0.85636 0 −4 −2 0 2 4 6 0 2 4 6

log2FC−Meta log2FC−Meta Supplemental Figure 2

analysis A 5000 B Meta-analysis 10 linear fitting Re-analysis meta-analysis 4000 re-analysis

5 3000

2000 DE DE genes 10 0 log 1000 R2 = 0.9607 2 0 −5 R = 0.9512 null 2 3 4 5 6 7 8 9 10 11 12 0 5 10 15 number of overlaps across studies consistency

C Meta-analysis D Re-analysis

Kowalczyk 0 5 4 23 31 20 20 55 8 11 77 0.81 Mann high 68 0.82 Grover 7 6 4 9 30 13 20 43 8 60 0.82 Lazare 83 17 0.92 0.87 Quere 7 9 5 11 24 23 36 78 204 0.49 Wahlestedt 313 41 31 625 207 46 38 0.71 Sun 35 98 81 186 311 208 381 2634 0.35 Norddahl Bersenev 329 122 112 45 23 0.76 Chambers 19 32 81 167 111 181 2573 0.27 Sun low 2622 176 277 170 74 34 0.3 Rossi 7 32 31 53 91 737 high 0.47 Maryanovich 1100 503 140 180 140 56 33 0.56 Wahlestedt 4 48 21 64 408 0.84 Chambers 1770 141 174 48 116 61 17 14 0.2 Flach 3 9 30 888 0.39 Kowalczyk 272 47 51 70 40 58 40 15 32 0.49 4 9 421 0.41 Beerman Grover 99 28 29 25 31 31 34 25 14 18 0.67 fraction of total overlap 20 155 0.82 fraction of total overlap Noda Flach 176 12 18 32 37 40 30 43 29 13 9 0.52 Kirshner 62 low 0.82 Kirshner 30 5 7 9 8 8 8 12 10 9 5 5 0.67 v er er v v vich Sun Sun man Noda Flach Flach Rossi Mann Quere Gr o Gr o Lazare an o Kirshner Kirshner y ow alczyk ow alczyk Norddahl ahlestedt ahlestedt Bee r Bersen e Chambers Chambers K K W W Ma r

E genes ≥ 4 studies 6000 142

220

4000 DE genes 2000

0 meta-analysis re-analysis Supplemental Figure 3 A B 30 signaling receptor activity 25/1020 regulation of cell population proliferation homodimerization activity 22/868 regulation of response to stimulus cytokine receptor activity 7/88 cell differentiation response to oxygen−containing compound protein dimerization activity 25/1343 20

ion binding 63/5302 regulation of response to external stimulus intracellular signal transduction carbohydrate binding 10/265 calcium ion binding 15/594 response to endogenous stimulus

FDR upregulated lymphocyte activation 2 transmembrane signaling receptor activity 18/863 10 enzyme linked receptor protein signaling pathway −log lipid binding 15/652 All genes aging signature response to external stimulus cell adhesion molecule binding 8/207 regulation of protein phosphorylation cell surface receptor signaling pathway 0 5 10 15 −log FDR cell−cell adhesion C 2 0 −log FDR 2 0 10 20 30 0 2.5 5 7.5 10 −log2FDR downregulated Fluid shear stress and atherosclerosis 9/140 degranulation 8/121 D Transcriptional misregulation in cancer 9/167 WT Chemical carcinogenesis 6/92 Complement and coagulation cascades 6/88 Young/Aged Glutathione metabolism 5/61 Size/singlets/Alive RA biosynthesis pathway 4/35 Cell surface interactions at the vascular wall 6/103 upregulated patways LSK Formation of Fibrin Clot (Clotting Cascade) 4/34 LT Lin- Signaling by Nuclear Receptors 6/135 HSC

2/39 IL−3, IL−5 and GM−CSF signaling

3/158 Cell adhesion molecules (CAMs)

3/118 Degradation of the ECM Young Cytokine−cytokine receptor 4/252 interaction 6/476 Neutrophil degranulation

5/262 Signaling by Interleukins

2/8 IL−35 Signalling

4/161 Jak−STAT signaling pathway

downregulated patways Aged 3/35 IL−2 family signaling

3/35 DNA replication Lin

8 6 4 2 0 C-KIT CD48 CD48 SCA-1 CD150 −log2FDR

E 0.5 40000 F Young Aged **** **** 0.4 DAPI 30000

0.3 FI/ cel l

merge M 20000 0.2 O RNA/cell (pg ) SY T 10000 0.1 SYTO

0.0 0 Young Aged Young Aged Supplemental Figure 4

A Size/singles/Alive/Lin- Size/singles/Alive/Lin- LT-HSC CMP MPP ST-HSC GMP MEP MPP LK GMP

LSK ST LT CMP HSC HSC MEP

Young Young

Aged Aged CD 16/32 C-KIT C-KIT norm. count norm. count CD48 SCA-1 CD150 SELP SCA-1 CD34 SELP

B

Size/singles/Alive

SELPlow

~30% ~30% LT - LSK high Lin HSC SELP

Aged Lin C-KIT CD48 CD150 CD48 SCA-1 CD150 SELP

C day 6 day 8 day 10 Selplow Selplkigh Selplow Selplhigh Selplow Selphigh Size/singles/Alive

My PBS MAC-1 CD16/32

TNF-α

LPS MAC-1 CD16/32 Supplemental Figure 5 A Size/singles/Alive Size/singles/Alive Size/singles/Alive WBC PLT Ery

EV

SELP OE GFP GFP GFP CD45.2 CD41 Ter119 B

Size/singles/Alive/ CD45.2+

80 EV EV SELP OE 60

40

Size/singles/Alive/ CD45.2+ 20

SELP OE 2 1 Frequency in bone marrow (% ) 0 A CD71 CD71 CD71

GFP Ter119 FSC-A 19+ ProE Ery Ery B Ery C er 1 T Supplemental Figure 6

Genes associated with GO terms with “membrane”:

Abca4,Abcb1a,Abcb1b,Acpp,Acsl4,Adgrg2,Alcam,Amotl2,Antxr2,Anxa2,Anxa6,Arhgap30,Arhgef28,Art4,B3galt1,Bmpr1a,Casp12,Cavin2, Cd200r4,Cd34,Cd37,Cd38,Cd63,Cd86,Cd9,Chrna7,Clca3a1,Cldn12,Clec1a,Clec1b,Clu,Cntn1,Col4a2,Cpne8,Csf2rb,Ctss,Cxcl16,Cyb561, Cyp26b1,Cysltr2,Cyyr1,Ddr1,Dennd5b,Dhrs3,Dnm3,Dsg2,Ebi3,Efna1,Ehd3,Enpp5,Evc,Exoc6b,Fap,Fhl1,Flt3,Gabra4,Gem,Ghr,Gm10419, Gpr183,Gstm2,Hpgds,Id2,Il12rb2,Il1rapl2,Itgb3,Jam2,Jun,Kcnip3,Kdr,Klrb1b,Klrb1c,Lamp2,Ldhd,Lgals1,Lpar6,Lpl,Lrrn1,Lsp1,Lsr,Lst1, Ly6e,Mamdc2,Mgst1,Mlec,Mmp14,Mmp2,Muc1,Muc13,Myo1e,Nckap1,Ndrg1,Neo1,Npdc1,Nrg4,Ntf3,Ocln,Osmr,Pdgfd,Perp,Pgr,Pla2g4a, Plcl1,Plek,Plscr1,Plscr2,Plscr4,Plxdc2,Ppp1r16b,Prcp,Prnp,Procr,Prtn3,Ptger4,Ptprk,Rab34,Ramp2,Rdh10,Rgn,Rhoj,S100a6,Sell,Selp, Sema7a,Sfrp1,Slamf1,Slc14a1,Slc28a2,Slc6a15,Stom,Stxbp4,Syk,Tacstd2,Tgm2,Thbd,Tm4sf1,Tm6sf1,Tmem176a,Tmem215,Tmem47, Tnfsf10,Trpc1,Trpc6,Vldlr,Vmp1,Zg16

Genes associated with GO terms with “inflammation”:

Bcl6,C4b,Camk1d,Casp12,Cd200r4,Chrna7,Ctss,Cyp26b1,Il15,Lpl,Nupr1,Pla2g4a,Ptger4,Selp,Sema7a,Slamf1,Tgm2,Zfp36

Genes associated with GO terms with “cell cycle”:

Abcb1a,Btg2,Fap,Fhl1,Gadd45g,Gem,Id2,Jun,Mcm5,Mcm7,Nupr1,Ptprk,Rassf4,Sfrp1,Stxbp4

Genes associated with GO terms with “histone”:

Bcl6,Dnmt3b,Egr1,Mllt3,Pla2g4a

Remaining Aging signature genes which were not found associted with search keyterms:

Abat,Aldh1a1,Ampd3,Arhgap29,Asb4,Aspa,Cytip,Dhx40,Ect2,Fhdc1,Fyb,Gda,Gipc2,Gpx3,Gstm1,Gstm7,Hnf4a,Klhl4,Mab21l2,Maf,Matn4, Mef2c,Meis2,Mmrn1,Mt1,Ndn,Nrk,Nt5c3,Oxr1,Pbx3,Pcdhb16,Pclo,Phactr1,Phf11d,Plac8,Pros1,Rbpjl,Rfc2,Rnase6,Rorb,Rorc,Runx1t1, Satb1,Sbspon,Sdpr,Serpinb6a,Serpinb8,Socs2,Sult1a1,Tbc1d8,Tc2n,Tdrd9,Tmem56,Tox,Trim47,Tsc22d1,Vwf,Wwtr1,Zfp334,Zswim5

DE expression upregulated downregulated Supplemental Figure 7

Aging Signature A B Genes found in aging signature (consistency >= 4) GeneGene A BGene C Z Show 10 entries Search:

Gene Average GSEA (continous) symbol Gene description Consistency LogFC SD

Selp , platelet 12 2.37 1.27 Gene A > Gene B > Gene C > ... > Gene Z Mt1 metallothionein 1 11 2.13 1.48 Nupr1 nuclear protein transcription regulator 1 11 1.80 0.86

: 1 2 3 n Clec1a C-type lectin domain family 1, member a 10 1.54 0.71

Plscr2 phospholipid scramblase 2 10 2.79 1.61

Enpp5 ectonucleotide 9 1.55 0.77 pyrophosphatase/phosphodiesterase 5

Gstm2 glutathione S-transferase, mu 2 9 2.26 1.25

Gene GeneA B/C Gene Z Itgb3 9 1.25 0.46 dGSE (discrete) Alcam 8 1.43 0.55 Cd38 CD38 antigen 8 1.57 0.88

Gene Gene description Consistency Average SD Gene A > Gene B = Gene C > ... > Gene Z symbol LogFC rank: 1 2 2 n Showing 1 to 10 of 220 entries Previous 1 2 3 4 5 … 22 Next

www.agingsignature.webhosting.rug.nl

AGING Dashboard SIGNATURE

Dashboard About Aging Signature

Search This website was developed as a webtool part of the studied entitled “A comprehensive transcriptome signature A list of the genes which comprise the Aging Signature. This list provides the gene symbols, gene description, of murine hematopoietic stem cell aging”. Here we aim to provide easy, intuitive and code-free access to the average logFC, standard deviation (SD) and number of studies which a certain gene was found to be differentially Aging Signature dings which were described in the study. expressed in 4 studies out of 12. The current version allows you to have an overview of the 220 AS genes or search for speci genes. Brie y, this dataset resulted from a systematic re-analysis of multiple (12) transcriptomic studies of young and Pyramid physiologically aged murine Hematopoietic Stem Cells (HSCs). From these independent studies we generated a Aging Signature list of the most robust genes which were present in such studies – termed the Aging Signature (AS) – but also a dGSE complete list of all differentially expressed genes – The Aging list (AL).

We wish to provide quick access to researchers which are interested in enquire if a speci c gene(s) are directly Aging List Aging List involved in transcriptomic changes in mHSCs. Furthermore, we also provide access to Enrichment analysis which aims to compare a list of genes with the Aging Signature (addressing the question “How close a certain list of Publications genes is related to physiological aging?”). A list with all genes which were ever reported to be differentially expressed in mHSC aging. Unlike the

All supplemental information can be found in the published manuscript and on our github. Aging Signature section, here were also provide information about genes with low reproducibility. Aging list

Publication overview

dGSE signature plot List of the studies that were used as source data from the development of the Aging Signature and Aging List. Discrete Gene Set Enrichment is an algorithm which compares different transcriptomic sets to the Aging Signature. In this section, users can provide a list of genes (use input) and it will be compared to the AS using 2 Publication overview different methods (Rank Score and Enrichment Score). After the analysis is done, Enrichment plots are displayed showing the user input together with the different studies which comprise the AS, giving an idea of how much the user input list is comparable with the Aging signature. 2 examples are provided as reference.

Search gene or multiple genes dGSE signature

Quick search tool which allows to enquire for speci gene(s). Search results return if the enquired gene is part of the Aging Signature or not. Multiple gene search is limited to 500 genes at a time.

Search

Search dGSE

Search single gene

Search multiple genes Search for gene ... Example lists

Search multiple genes Challen: description of the data

dGSE signature

* The search option is limited to a maximum number of 500 genes. Download data Maryanovich: description of the data * The search option is limited to a maximum number of 500 genes.

dGSE signature Genes search result

GENE(S) IN AGING SIGNATURE (AS) GENE(S) IN AGING LIST (AL) Download data 3 131

GENE(S) NOT IN SIGNATURE GENE(S) NOT FOUND 266 1

Gene(s) in annotation, in Aging Signature (AS) Genes in annotation, not in signature

Ampd3 Nt5c3 Ctss Comt Slc1a5 Trappc6a Rmnd5a ......

dGSE signature

Gene(s) in annotation, in Aging List (AL) Genes not found

Gmpr Ube2c Ssr4 Prkar2b Ctse ...... Gm4604///Rpl36

dGSE signature