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 genes 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 protein 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 Platelet 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 Gene Z Show 10 entries Search:
Gene Average GSEA (continous) symbol Gene description Consistency LogFC SD
Selp selectin, 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
rank: 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 integrin beta 3 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
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