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1 DOI: /n3296 Supplmntry Figur 1 PN2 PN5 Exprssion o Tt trnsripts uring mryos vlopmnt. x3 x7 x9 x4 2lls 4lls Blstoyst 5C 5C 5C 5C x3 x1 x4 Mrg (5C/5C, PI) x6 x9 x8 x ng ntioy C ntioy C 200ng 1:2 ilution 12.5ng 4 1: ilution + nulosis 6 pg + nulosis Supplmntry Figur 1 () Tt3 stining in zygots shows n umultion o Tt3 in th ptrnl pronulus rom PN2 n grul nrihmnt rom to PN5. Tt3 protin is not ttl ollowing th 2-ll stg. Rprsnttiv imgs r shown (rplits init on th igur). () Exprssion o Tt1/2/3 in mous primplnttion mryos (s on 39 ). () 5C n 5C stinings in n zygots. Not tht in th rly zygot 5C (n 5C) is osrv in th mtrnl pronulus only (s lso Supplmntry Fig. 2,). stin with PI. Rprsnttiv imgs r shown (rplits init on th igur). () Dot lot nlysis o C-, - n - ontining PCR prouts (mplii with CTP, 5mCTP n 5hmCTP, rsptivly) to ssss th snsitivity n ross-rtivity o nti- n nti- ntiois. ntioy shows out 10,000 ol grtr snsitivity thn th ntioy. () Comptition ssy to ssss spiiity o ntioy using gnomi rom 3T3 lls ovrxprssing Tt1 or Tt3 protin. Comptitor nulosis (200-ol xss) r 5-mthyloxyytiin () n 5-hyroxymthyloxyytiin ()., ml pronulus;, ml pronulus; 5C, 5ormylytosin; 5C, 5roxylytosin;, 4,6 -imiino-2-phnylinol; PI, propiium ioi; RPKM, rs pr kilos o trnsript pr million mpp rs. (Sl rs, 5um) Mmilln Pulishrs Limit. All rights rsrv.
2 -PCR prout tmplt Enzym mM 10mM 25mM ynmis (ontrol vs ) ynmis () vs ynmis (ontrol) 40ng Dilution 1:4 ttion G1 S G2 G1 S G2 5C (5C, PI) 5C (5C, PI) Emryo vlopmnt tr Tt3 inhiition 2lls 4-8lls lstoysts 2lls 2lls 4lls 4lls morul morul 24 hp 48 hp 72 hp 96 hp Supplmntry Figur 2 () in vitro tivity ssy in th prsn o. Convrsion o to is ssss y ot lot. () Dynmis o n in mous zygots trt with. Not th initil ispprn o ptrnl spit th sn o in trt zygots. Th umultion o in -trt zygots is ttl only in th lt PN stgs n orrlts with th pprn o in ontrol zygots (n=192 zygots). 5C (n=8 n n=9 ontrol zygots; n=8 n n=6 -trt zygots) () n 5C (n=6 n n=8 ontrol zygots; n=8 n n=7 -trt zygots) () stinings in -trt n zygots. Quntiition is rprsnt s rtio twn th pronulr signls (pt/mt). Not tht in th rly zygot 5C (n 5C) is osrv in th mtrnl pronulus only (s lso Supplmntry Figur 1). (Sl rs, 5um.) () stining in pr-implnttion mryos riv rom in vitro rtilistion in th prsn o. is not ttl t 2-ll stg ollowing trtmnt whih is in grmnt with th sn o ttl Tt3 protin t this stg (Supplmntry Fig. 1). is grully ttl rom 4-ll stg onwrs. (Sl rs, 10um.) () Lk o zygoti ( trtmnt uring IVF) os not t th vlopmntl potntil o mous mryos (n>20)., ml pronulus;, ml pronulus; 5C, 5ormylytosin; 5C, 5roxylytosin;, imthyloxllyl glyin; PI, propiium ioi. Sttistil nlysis ws rri out using Stunt s t-tst (two-si). Error rs init s.. **, p<0.01; ***, p< Mmilln Pulishrs Limit. All rights rsrv.
3 Supplmntry Figur 3 H3K27m3 H3K9m2 DFX H3K36m3 PI H3K4m2 DFX PI -PCR prout tmplt Ptrnl pronuli xtrtion Lin1 rptitiv lmnts Enzym DFX mM 10mM 20mM Bisulit squning on ptrnl pronuli only 120ng Dilution 1:4 ttion Lin1 rptitiv lmnts p = % 88.3% 5 UTR ORF1 ORF2 3 UTR (A) n pg + pg 245p CpG p= % 46.2% p= % 31% 77.5% 80.2% Lin1 rptitiv lmnts Zygots -5 Zygots -5 p= % 73.1% p= % 54.3% 2lls (30 hp) Supplmntry Figur 3 () Histon moiition symmtry is mintin in -trt zygots s ssss y stining with spii ntiois or H3K4m2 (n=5 zygots), H3K9m2 (n=5 zygots), n H3K36m3 (n=5 zygots) n H3K27m3 (n=8 zygots). Rprsnttiv imgs r shown. () in vitro tivity ssy in th prsn o F 2+ hltor roxmin (DFX). Convrsion o to is ssss y ot lot. () Inhiition o Tt tivity using DFX ls to rs in ptrnl without ting th loss o ptrnl. Vlus r rprsnt s rtio twn th signls in prntl pronuli (pt/mt). For stining, n=8 ontrol n n=8 DFX-trt zygots; or stining, n=8 ontrol n n=8 DFXtrt zygots. Sttistil nlysis ws rri out using Stunt s t-tst (two-si). Error rs init s.. () Bisulphit squning nlysis o th 5 UTR o Lin1 rptitiv lmnts (L1M_T n L1M_G sutyps) initing th prntg o mthylt n hyroxymthylt CpG inulotis in zygots without polr ois (n=22 n n=20 mplii lons riv rom ontrol n -trt zygots, rsptivly t, Tt3 WT Tt3 mt-/pt+ Zygots -5 10hp; 2 inpnnt iologil rplits r shown) n in 2-ll mryos (n=24 n n=21 mplii lons riv rom ontrol n -trt mryos, rsptivly). Not tht isulphit squning os not istinguish twn n. () Bisulphit squning nlysis o th 5 UTR o Lin1 rptitiv lmnts (L1M_T n L1M_G sutyps) on th ptrnl pronuli o ontrol n -trt zygots (lt -, 7-10hp) xtrt y miromnipultion (n=44 n n=42 ptrnl pronuli xtrt rom ontrol n -trt zygots, rsptivly; 2 inpnnt iologil rplits r shown). () Bisulphit squning nlysis o Lin1 rptitiv lmnts (L1M_T n L1M_G sutyps) in wil-typ (Tt3 WT ) n Tt3 mtrnlly plt (Tt3 mt-/pt+ ) zygots rovr t, 9hp (n=31 n n=31 mplii lons, rsptivly). For isulphit xprimnts, p vlus wr lult using Mnn-Whitny U-tst. **, p<0.01., ml pronulus;, ml pronulus;, imthyloxllyl glyin; DFX, roxmin; PI, propiium ioi. (Sl rs, 5um.) Mmilln Pulishrs Limit. All rights rsrv.
4 LOD LOQ C 100 mol 500 mol G 100 mol 500 mol 5 mol (14) 5 mol (11) Prthnots 7hrs post-tivtion Mrg EU puls (EU, ) Prthnots 10hrs post-tivtion Mrg EU puls (EU, ) 2.05% (LC/MS) 2.29% 2.51% Aphiiolin Prthnots (LC/MS) (LC/MS) 3.143% 2.072% 2.93% 0.032% 0.938% 0.775% 1.60% 1.48% n.. n.. (<0.016%) (<0.008%) EU Mrg (EU, ) g Aphiiolin Aphiiolin (DMSO) h (LC/MS) i ynmis (ontrol vs Tt3 mt-/pt+ ) j Lin1 rptitiv lmnts p= p 5 UTR ORF1 ORF2 3 UTR (A) n pg + pg CpG 32.9% 3.22% 26.5% 1.83% 2.02% x4 AzC Zygots -5 (11 hp) Supplmntry Figur 4 () Tl summrising limit o ttion (LOD), limit o quntiition (LOQ) n signl-to-nois (S/N) rtio (in rkts) o LOQ pk or nlys nulosis in our LC/MS mtho. () Prthnogntilly tivt ooyts wr inut or short puls with EU post-tivtion to ontrol or th ompltion o S-phs. (n=4 prthnots.) Rprsnttiv imgs r shown. (Sl rs, 5um.) () LC/MS quntiition o /G rtio in MII ooyts n prthnogntilly tivt ooyts lok or rplition, showing rltiv stility o gnom-wi mthyltion on th mtrnl gnom (2 iologil rplits n 2 thnil rplits, 100 mryos (or ooyts) us pr msurmnt). () Clultion o ptrnl lvls s on th ssumption tht signl on th mtrnl pronulus rmins stl in th zygot (oring to msurmnts in Fig. 2). Error rs init s.. () LC/MS msurmnt o /G n / G rtio in sprm, ontrol n -trt zygots (with polr ois) onirming rsults shown in Fig. 2. Eh vlu rprsnts th vrg o 2 inpnnt xprimnts with 2 thnil rplits; 100 zygots us pr msurmnt. For pks low quntiition limit, n ovrstimtion o /G rtio is lult s on th limit o ttion o. () Inhiition o rplition in phiiolin-trt zygots ssss y EU inorportion. (g) n stining in ontrol n phiiolin-trt zygots. Loss o n inrs in signls our inpnntly o rplition. Eh t point rprsnts n inpnnt zygot (n=9 ontrol n n=12 phiiolin-trt zygots). Sttistil nlysis ws rri out using Stunt s t-tst (two-si). Error rs init s.. (h) LC/ MS msurmnt o /G rtio in phiiolin-trt zygots (with polr ois). Eh vlu rprsnts th vrg o 2 inpnnt xprimnts with 2 thnil rplits. 100 zygots us pr msurmnt. (i) kintis in WT n Tt3 mtrnlly plt zygots (Tt3 mt-/pt+ ). (j) Bisulphit squning o th 5 UTR o Lin1 rptitiv lmnts (L1M_T n L1M_G sutyps) initing th prntg o moii CpG inulotis in ontrol (DMSO) n zc-trt zygots tr rmovl o oth polr ois (, 9hp). p vlu ws lult using Mnn- Whitny U-tst., ml pronulus;, ml pronulus; EU, 5-thynyl oxyuriin. (Sl rs, 5um.) Mmilln Pulishrs Limit. All rights rsrv.
![) () Anlysis o moiitions in zygots riv rom in vitro rtilistion o [Dnmt3L-/-] ooyts y](/docs-images/84/89136613/images/5-17.jpg "wil-typ sprm. Quntiition is rprsnt s signl intnsity in prntl pronuli.")
5 S U P P L E M E N TA R Y I N F O R M AT I O N Supplmntry Figur 5 Dnmt3 Dnmt3L (, ) PN2 WT Erly Lt Dnmt3L-/- WT ESC WT-ESC TKO ESC (, ) Mtrnl PN () printromri htrohromtin TKO-ESC np np Ptrnl PN () - pr-xtrtion x5 Aphiiolin EU puls n hp (lt ) x4 6.5 hp (lt ) x3 x3 EU puls 5.5 hp (rly ) XRCC1 XRCC1 h g + pr-xtrtion 5.5 hp (rly ) np Supplmntry Figur 5 () Dnmt3 n Dnmt3L r ttl in oth zygoti pronuli rom rly. ountr-stin with. Rprsnttiv imgs r shown (n=9 zygots) (Sl rs, 5um.) () Anlysis o moiitions in zygots riv rom in vitro rtilistion o [Dnmt3L-/-] ooyts y wil-typ sprm. Quntiition is rprsnt s signl intnsity in prntl pronuli. Eh t point rprsnts n inpnnt zygot. (n=11 WT n n=11 KO zygots). Sttistil nlysis ws rri out using Stunt s t-tst (two-si). Error rs init s.. ***, p< (Sl rs, 5um.) () Immunostining o wil-typ (WT) or tripl Dnmt knokout [Dnmt1-/- Dnmt3-/- Dnmt3-/-] (TKO) ES lls with n ntiois. (Sl rs, 15um.) () n stining ollowing SCNT into nult ooyt using WT (lt pnl) or TKO (right pnl) ES lls 14 hours post-tivtion. Dtil o mi-stion shows tht printri rgions (yllow rrows) gin in WT-ESC n TKO-ESC psuopronuli ut r prott rom novo hyroxymthyltion. ountrstin y propiium ioi. (Sl rs, 15um.) () Conol stions o ptrnl n mtrnl pronuli in mous zygots o-stin with n. Not th symmtry in / moiitions twn prntl pronuli. ountrstin y propiium ioi. (Sl rs, 5um). () Totl (-pr-xtrtion) n hromtin-oun (+Triton pr-xtrtion) XRCC1 in ontrol n -trt zygots rlting th tivtion o BER pthwy inpnntly o umultion. ountr-stin with. Rprsnttiv imgs r shown. (g) LC/MS quntiition o isotop-lll C (C*) rtio ovr G in rly pr-rplitiv zygots n zygots trt with phiiolin (5.5hp). Exprimnt rri out in thnil n iologil uplits. (h) Rplition kintis visulis y EU puls in pr-rplitiv (5.5hp) n post-rplitiv (6.5hp) zygots. Rprsnttiv imgs r shown (n=8). (Sl rs, 5um.), ml pronulus;, ml pronulus;, imthyloxllyl glyin; hp, hours post-rtilistion, n.., non-tt Mmilln Pulishrs Limit. All rights rsrv.
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