Figure S1. MUT-16 localization in L4 hermaphrodite, adult hermaphrodite, and adult male germlines. MUT-16 DAPI. Phillips et al. S-1. male.
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1 Supplementary Material for Phillips et al. Figure S1. MUT-16 localization in L4 hermaphrodite, adult hermaphrodite, and adult male germlines. Figure S2. Mutator foci and P granules localize independently of one another. Figure S3. Localization of MUT-16 and MUT-7 in CSR-1/WAGO class 22G mutants. Figure S4. ClustalW alignment of mut-16 in Caenorhabditis species. Figure S5. Characterization of HA::ego-1 FLAG::rrf-1 transgene. Table S1. List of genes tested by RNAi for effect on Mutator foci. Table S2. Homology and amino acid composition of mut-16 orthologs. Table S3. Classification of low and high sirna yielding genes (separate file). Table S4. sirna reads from high sirna yielding transposons. Table S5. Strains used. Table S6. Primers used. L4 3 day adult Figure S1. MUT-16 localization in L4 hermaphrodite, adult hermaphrodite, and adult male germlines. male MUT-16 DAPI Phillips et al. S-1
2 A B C MUT- 16 association with nuclear pores MUT- 16 nuclear pores DAPI merge D MUT- 7 association with P granules MUT- 7 PGL- 1 DAPI merge P granule components localizes independently of mutator proteins wild- type mut- 16 (pk710) rde- 2 (pk1657) mut- 2 (r459) mut- 7 (pk720) mut- 15 (tm1358) MUT- 16 localizes independently of P granule components control RNAi MUT- 16 PGL- 1 mut- 14 (pk738) glh- 1 RNAi MUT- 16 PGL- 1 pgl- 1 RNAi MUT- 16 PGL- 1 Figure S2. Mutator foci and P granules localize independently of one another. Phillips et al. S-2
3 MUT-16::GFP MUT-7::GFP rrf-1(nec1) ego-1(om97) drh-3 (tm1217) ekl-1 (ok1197) Figure S3. Localization of MUT-16 and MUT-7 in CSR-1/WAGO class 22G mutants. Phillips et al. S-3
4 Figure S4. ClustalW alignment of mut-16 in Caenorhabditis species. briggsae_mut-16 MSGLCENSFEQRTVLIGSHLGISATGLVMTSQEDDYPDDFDITTSEQ------GDADDIQ 54 remanei_mut MTEVNDEDYPEELDLSHTTEENEGIVGSVPSDE 33 brenneri_mut MSHSDDDYPELDVSTDGNEN----ALTAADGY 28 elegans_mut MSESDDDYPELDISDQYIDPLGIVVGPPPASY 32 japonica_mut MSDESYADLDLDSSYNEE EQPVQ 23.::.*.: : briggsae_mut-16 NHCLGAPPISDFDSNEEQ DSENELEDSYDSGSEPFDVDGYDYTT-EPL 101 remanei_mut-16 MHFIGAPPGSDSEVDEEYR QYQRDPYSDYSSSDNEFDCDDYLKNVPREL 82 brenneri_mut-16 GEVVGAPPSSDVESDESAYHTPSSEENVIRQYEYNECDLYDSGSEEFDVEGYENEQLADL 88 elegans_mut-16 TETDREETPMQNRTEDDTN------SYGNSSGEHDYDSYLDSGDDDFDVDAYYANDNMDE 86 japonica_mut-16 NHVIGPAPTSSEDDEMDTR SVDSYMSGESDYDLENATPYIEPSVEELAKLD : : briggsae_mut-16 PAMPDD---LVGNGAQN--YADMNDFGGDPRELLPIFFTSTLAAHPLRLKEGYDPEELDK 156 remanei_mut-16 PELTND---LIVSGSDNTRLQELQDLG-DPVEFLKVFLTASMMGQPLKVKDGFDPEDLDK 138 brenneri_mut-16 HIHDMSSYVDVGNEQESNAAHNFQEFEDSNKEIFQVLLPSAMIASIEHMTIGFEPSVIND 148 elegans_mut-16 PPPETIPDNLIQNVIGRNDNADYDFSDASNPEIMKLFLSSSLLSNPLKFRSGYSSDELDD 146 japonica_mut-16 ENLEPELPEHLIAAENYSDDIYASNSNEYVETVIKYFLPAAICSTPLLLRNGFSPDEVDK 134 :..: ::.:::.. *:... ::. briggsae_mut-16 CLRENMGFTLPVVATILLGSHITDGLPNDSKTEYAIALANKNWLTRNGDKFLPIFPESEK 216 remanei_mut-16 CCSELMGITLTGVASVLLGAELQN-IPNRSKTEMAFALANRGWLRAVNSKFYPVIPETEN 197 brenneri_mut-16 CLKD-VGYCLEVVAMMLLGAEKFATIPRAP-VDWCHALKDANHLLFENGKFYPRVPESLR 206 elegans_mut-16 CLKSCMGYSLQVTAVLLLPPEIISQLPNDSKTEHAHALVRGGWLQSKEGAFFPVISDSER 206 japonica_mut-16 CLTDEGQISLEATAYILLGARIVENLPNKSKTEFCHALVKAGWLTEKQGKFFPVLPESEK 194 *. *.* :**.. :*...:. **. *. * *..::. briggsae_mut-16 EFITSLIEGSELLASKEETKKKEAQVFPSMEKEIRAHQTFNMIVELLQIVRDELRQRKVP 276 remanei_mut-16 DFVLSLIEGAERLQHLDENKKREAEHYPSAEKEINALISFNMIVELMDAVREEYRIMLLP 257 brenneri_mut-16 AHCTSLIEGASRLSQFEEKKKKEQETYKSEEFELRVLYTFNMIAELLNAVRDEQRQLKIG 266 elegans_mut-16 ETVVSLMNGSEEQHKRQERKKKEADTFESEEKEIRTLLTFNMIAELLMAVRNEYSIRSVK 266 japonica_mut-16 EFILELIDGAENTKYYEERKKKEAEQYPSEEAEIRAHITFNFICELLSAVQRELQQNKIK 254.*::*:. :* **:* : : * * *:.. :**:* **: *: * : briggsae_mut-16 YQNIMFAFNEMAKGKKHAHVFEKYSKQLELDS-TMEWNTKWFNQYTNRSTLKKFVQMSKF 335 remanei_mut-16 YQTLSNGYLDMVTGKKYPEIHKKYFNKLNLDE-KKVWNSEWFNAFTGRSTLKKFVQMARF 316 brenneri_mut-16 YSHLMNAYQAMVEGLKYHDIFKRYKGTLSLNDPKKLWNSQWFNEYTGRSSLMKFIHMARF 326 elegans_mut-16 YQILSTAYTNMVTGAAHANIFRKYKDILQLDP-EKLWNNDWFKEYTNRGTLKKFLTTARF 325 japonica_mut-16 YQTLSTEFEKLVSGQKNANLFSKYKHLLDLES-DAKWDSEWFKKYTNRSSLKKFVTMPKF 313 *. : : :. *.:. :* *.*: *:..**: :*.*.:* **:.:* briggsae_mut-16 SEIVVSNMK--DSPFYFRADDEG-DRPVKMFTDDDIKAVKDKWMSGNERNKNFGS--NYE 390 remanei_mut-16 AEIVVVENP--TSQFCFRADSE--NRPVRMFTANDIKDVEEKWSSGNEKNKNFGR--NYG 370 brenneri_mut-16 SEIAVTNTN--PRTYYFRADDDVPQRDVKLFIKKDFDEVREKWRSGNQANRNFGR--NYG 382 elegans_mut-16 SEIVVSQANGKTVELYFRADDEG-NRPVVLFTDEHIADVRNKWKTGNQRNQNYGSQGNYR 384 japonica_mut-16 AAIVADQTS----EFRFRTDNVH------LFTNEDLDEVRQRWR : *.. : **:*. :*..: *.::* briggsae_mut-16 RSGQRGDRGGPSRYQQQPKKVIEQDPNYRSSTFNGGISGA-ANDDGSLEPSSSSRTYDNQ 449 remanei_mut-16 GQGQRGLPRGAS---QQPKRKIEQDPNYRSSTFAGGIN-D-DDDDGSLMPSTSARENQAG 425 brenneri_mut-16 AGGQRGEGRGPS---QQKKKTIEQDPNYKSARFNGLMD---GVDDGSLEPSTSTRYNRE- 435 elegans_mut-16 AGGQRSDDRRGP---QQRRNVIVPDPNYQPSTFAGGISNN-ADDDGSLQPTTSSHFNRNT 440 japonica_mut-16 RPAARADRRWQSGSQNAKSRENVPDPDYRPAAVSGGISALDEDDDGSLRPTTSSNHRNRH 407. *.. :. **:*:.:. * :. ***** *::*:. briggsae_mut-16 PSSRTR CHRSPTPSPERRRENPVAERSTIRDESTQRRSESPPPVTR 495 remanei_mut-16 PSSSSQPV YGRRSPSPTVRNQRARSPSQESRNSNTVPQSSYRPIDPFAG 474 brenneri_mut RSPPRPIRNRSLSPRENRSPIPARSPSPFDEPTSQAQE 473 elegans_mut-16 DRSTSRP PRAPTSPVNRVMETDPLMGQGTSSGAPQRSAIP-NPFGG 485 japonica_mut-16 HSPTATIRDSSRTRPTTSSHPARSRSPSITTKTTRTRRDSSENPPPEPLNMEPAANPFGG 467 *:..... briggsae_mut-16 EVFDPFGGGPPLNQKTEDNRGNNRGRGFGNGNFSS remanei_mut-16 AMVHPISAGVNGNRNGQGGYETRRLQTFGASSSESDEVSEGCYSDDDPEDQKRKTIERNE 534 brenneri_mut-16 QAARPSSPE elegans_mut-16 APALSRSTITNGNRGPSYGDRGERVQDVGDTTSDSEITSEGSYSD japonica_mut-16 PLPPPARAEHRPAHQPPPPNFPRTADYAGYSSTEDDITSDGSYTD Phillips et al. S-4
5 briggsae_mut LAKPVARIDHIGDTSSETSSRFNT AYSPPRRRTPSPKPRRNV 572 remanei_mut-16 KRLRKEAREMERRKKHKAEVHVPPVTRFQTNPFYKHKSARAPEPANPRASTAVEQEEREE 594 brenneri_mut SQSDPPQRVALSDPFGGR 500 elegans_mut EDPEQKEIKRQRRKDKLKKKQERELRSREKHTKSKQQPPSKIETRFNTYKKKSES 585 japonica_mut EKSEEKERRRDDRQKKLAKKQKKLME RQNAPPKPPPDNSRNRFRVSS 559. briggsae_mut-16 SAEPEIPSPQPQPTQNQTPSPFLGQESPPS---IDNDRMDDYMPPPPAAPVVRQLPLAVS 629 remanei_mut-16 LVVPPRQSPFDAPSALAPAPAAPAPVSQPSNSDAQVNAHEDVLPPPPAAVAANTLPLFVS 654 brenneri_mut-16 PVQRREETNFGGSSDNQLTRDNYGR NRAGEVRRIDVHLI 539 elegans_mut-16 SATDTSNTPPVDTVNVALPTPVVESSSTTAAPSIPVSTRPEVVVPPENPAPLREVGNFYS 645 japonica_mut-16 FAPVPESPTPPPPAAPAAPPPLQETRNDPD PPPSLSGQIPLTIS : briggsae_mut-16 SSRHDA-EHMTAQVPYRPAEGVLERNKAEEEKKKRRQEIDDVRIESENRQPPTQHPFLTS 688 remanei_mut-16 SSTHDQ-DRCSAQMPYKPAEGVLERNKQEEAERLRREQLSEMRMRSGFNQVPASHPWMSA 713 brenneri_mut-16 GETSSE-GEITSDGSYSNED-----EDTKQAKRERRLAVKKKKDEREMR elegans_mut-16 KSNHDE-DRRNVQLPFTPADTHKPIKVAPKEPVRNPLLKERPSANGFINRRLPSHPAPPP 704 japonica_mut-16 ESRFDEEDQARGSIPFRPMPTSRPVEDERAAPVPPVPAPQPRTRVVHPDIARRQRDQNES : :. briggsae_mut-16 S------RAKPTTSRNPEQGYSDPVTTNGFARRNVAQPTYAATMAAQAPPAPPAPAPQPS 742 remanei_mut-16 ASKPGPSQPQAYEEPAPTKGFGTTLPPTVQRSVPQPPPQQAPQMQQQQEPIYSTPFVETV 773 brenneri_mut RRKQHEENRNKPIEQTRFSVSQFKKKTPQNAPRSPERRRQESPLPFEDEPTTS 635 elegans_mut-16 VN-----QSQPANQPMQTAVYQNSHPGAPYIPQQPTYQPQLPVQQPQPHQYAPQPIHHQQ 759 japonica_mut-16 VSRPMEPGVPGGFRGARKVEPAQAPPPPPPQTAPPPQPPRQPHQQPPPPLHNYQQFSDNR briggsae_mut-16 PVP PPVFPLHAPMQAPLQVPVSRTNSVIKIILKPSILE 780 remanei_mut-16 SLSSLIVLNNSLGFQYQGRNRQGMAPQNYAQPAPQNYAQPAPQNYAQPAPSQQQYPQHQN 833 brenneri_mut-16 TEP LPPVELPVEEPTPVQTTTADSDDEPLPVRPQ 669 elegans_mut-16 PIHQP MHGQQYPPVNQQQPIYQQPAPQYPPYNSIQN 795 japonica_mut-16 RGSQDYG GMNGRPESPPPIPEQQHHYQAPEPQYPHYQQQPN : briggsae_mut-16 EIDKEGGHLNRGYPLPPQQYPPQNQYQPPLQQQIQ----QPNYQYPQQNQYQQGPPPPQY 836 remanei_mut-16 LVQQNPPLFPDHSASQPQYQPMQQQYQQPLPQNPYPIGSQSSSTLPQQQPYHHQNQDTYR 893 brenneri_mut-16 ATNKAPAVVTDTMTN ALSHQAQVPYR 695 elegans_mut-16 NPQHGPSPFNYSQVPQPAYNHVGQQPSHMSNQPHINQNGYQNSYNPNQGPTSSDPNYGCN 855 japonica_mut-16 PYHERNNQPNRQFAPQPNNQPYSMGEPEQPREYNMPNYYNPNYQPPPPPPQHQQQPVYGQ briggsae_mut-16 PMETPQYNPTPPPPPRAEYSSNYPPPQNQMNR-----PPQQSYQDQQYSRNQYSSNGNWS 891 remanei_mut-16 PTSRNHANPQHMDNQYSSMRDAMRPPVPRNDMNYDSIPSNSLHQIPPMNQPQYSSQQRQY 953 brenneri_mut-16 PSALDVQKKKMEQEEKAKKASMN AVKKYGADAVLAYHPALFSQNSGPG 743 elegans_mut-16 PQFNHYGSRSVYHEDHSSQRRRSPDQFPPNPP---EYDPHGNFKLADYERDRMTVGYSQN 912 japonica_mut-16 NYHDPPRESRSSHFNNFQQPPVQSAPPPPHTSRNSHHQPFDHYFPSGNGNAHYNSNNGYN briggsae_mut-16 NPAYPQQPRSPPLPNGPPQDYNRWRDRDPPPRQPPPATYGRRDVGRPS remanei_mut-16 NDVYP--PVNPYNNRVPSTDFNDRRIRNEQWHQVQPGSYDPYGRSQNPGPGSSYNPRGEE 1011 brenneri_mut-16 PSSSPLPPPQAYIEPEPPSGFGNSQ elegans_mut-16 PHQFDHHGSHMPHQSQP-QGYDNFNGNSAPYFNKNGGQSNHQPEAQRS japonica_mut LPFANRQQQYHDQPPPPHNNDRWNPYATARRPPPATTGRGVSLLS *... briggsae_mut-16 --LSTFSLLAEEDDRRAAPQRPDVGRMRDIIMSIAYNCRTKGCQLDKERLKYEVCQSRFQ 997 remanei_mut-16 NCASFFGRLRKAAAASGGGESEEVKQIKIEIQRIVFDYASENRELTLSELKANLVRKMRH 1071 brenneri_mut elegans_mut FSVLSSNRQPSNRELIFQDGIEKELRDIILRYRSMNLTVLTVQELRTEVSRRPAI 1014 japonica_mut SHPRDSTQLSAEEDRRIQRGRMSVMRYMEDCERSRSQVTGYDLRSAQHNGEVH 982 briggsae_mut-16 QHFPGGPEWFDFTSFIRNELRGTMEVRGNENGAVWYELLRN remanei_mut-16 LQF------FDVHEFIQTYLRNQVVIVNNGPYGPVVRPT brenneri_mut RFVDYIKRFFV elegans_mut-16 PRY------IDIVQYIRDSSSVAIVERGDIEPYVVLKDDIRN japonica_mut-16 IGGN-----ENIIAFIRRYMSAIVGIGFGRDANGQEIDIFYVIQD :*: Phillips et al. S-5
6 A Somatic RNAi Fertility lin-29 nhr-23 wild-type ego-1 rrf ego-1 rrf-1;; HA::ego-1 FLAG::rrf B EGO-1 PGL-1 merge RRF-1 PGL-1 merge Figure S5. Characterization of HA::ego-1 FLAG::rrf-1 transgene. Phillips et al. S-6
7 Supplemental Figure Legends Figure S1. MUT-16 localization in L4 hermaphrodite, adult hermaphrodite, and adult male germlines. MUT-16 foci are present in the transition zone region of L4 hermaphrodite, three day old adult hermaphrodite, and adult male dissected germlines stained with DAPI (blue) and anti-gfp (recognizing MUT-16::GFP). All images are projections of 3D images following deconvolution. Scale bars represent 5 µm. Figure S2. Mutator foci and P granules localize independently of one another. (A) MUT-16 (red) associates with nuclear pores (immunostaining with mab414 - green) in C. elegans germlines. Images are single focal planes following taken from a 3D image following deconvolution. (B) MUT-7 (red) costained with P granules (immunostaining with PGL-1 antibody K76 green) at the diplotene stage of meiosis. Note that some P granules and Mutator foci have detached from the nuclear periphery and are now visible in the cytoplasm. Images are projections of 3D images following deconvolution. (C) MUT-16 (red) and PGL-1 (green) expression in adult C. elegans germlines feeding on E. coli expressing control (L4440), glh-1 and pgl-1 dsrna. PGL-1 and MUT-16 were visualized a PGL-1::RFP and MUT-16::GFP strain. Images are single focal planes from dissected (L4440 and glh-1) and intact (pgl-1) adult C. elegans. (D) Wild-type and mutator mutants were dissected and stained with DAPI (blue) and antibodies against PGL-1 (green). All images are projections of 3D images following deconvolution. Scale bars represent 5 µm. Figure S3. Localization of MUT-16 and MUT-7 in CSR-1/WAGO class 22G mutants. Balanced rrf-1(nec1) ego-1(om97), drh-3(tm1217); or ekl-1(ok1197) were introduced into the MUT-16::GFP or MUT-7::GFP strains. Homozygous rrf-1(nec1) ego-1(om97), drh-3(tm1217); or ekl-1(ok1197) animals were selected from heterozygous parents, and were subsequently dissected and stained with DAPI (blue) and anti- GFP (red). All images are projections of 3D images following deconvolution. Scale bars represent 5 µm. Figure S4. ClustalW alignment of mut-16 in Caenorhabditis species. Glutamines are highlighted in yellow, asparagines are highlighted in green, and prolines are highlighted in blue. Figure S5. Characterization of HA::EGO-1 FLAG::RRF-1 transgene. (A) Phenotypes of ego-1 rrf-1 mutant worms in the presence and absence of a rescuing transgene. Fertility scored as presence of viable progeny (+++) or complete sterility (-). lin-29 RNAi scored as 100% vulval bursting (+++) or 100% viable adults (-). nhr-23 RNAi scored as 100% larval arrest (+++) or 100% viable adults (-). (B) HA::EGO-1 (red, anti-ha immunostaining top panels) or FLAG::RRF-1 (red, anti-flag immunostaining bottom panels) costained with PGL-1 (green, anti-pgl-1(k76) immunostaining). DNA is counterstained with DAPI (blue, in merge). All images are projections of 3D images following deconvolution. Scale bars represent 5 µm. Phillips et al. S-7
8 Gene Description sirna Pathway +/- mutator foci L4440 negative control + mut-16 Mutator - Q/N rich domain WAGO-class 22G, ERGO-1-class 26G - mut-7 Mutator - 3'-5' exonuclease WAGO-class 22G, ERGO-1-class 26G - mut-2 Mutator - nucleotidyl transferase WAGO-class 22G, ERGO-1-class 26G + mut-15 Mutator - novel probable WAGO-class 22G + ergo-1 Argonaute ERGO-1-class 26G + dcr-1 DICER RNase III exo-sirna, ERGO-1 and ALG-3/4-class 26G + rde-4 dsrna binding protein exo-sirna, ERGO-1 and ALG-3/4-class 26G + ekl-1 Tudor domain WAGO-class 22G,CSR-class 22G + csr-1 Argonaute CSR-class 22G + cde-1 nucleotidyl transferase CSR-class 22G + rde-1 Argonaute exo-sirna + drh-1 DEAD box helicase exo-sirna + rsd-2 Required for sirna spreading + ppw-2 Argonaute + sago-1 Argonaute + Table S1. RNAi of each of the indicated genes was performed using the MUT-7::GFP strain, and scored for the presence or absence of Mutator foci. Negative (L4440 empty vector) and positive (mut-16 and mut-7) controls are included. Phillips et al. S-8
9 Table S2. Homology and amino acid composition of mut-16 orthologs BLAST results using N-terminal (1-530) or C-terminal ( ) regions of C. elegans mut-16 mut-16 (N-term.) % Coverage % Identity % Positive C. briggsae (29-557) 100% 42% 56% C. remanei (6-519) 99% 40% 56% C. brenneri (1-556) 99% 38% 54% mut-16 (C-term.) % Coverage % Identity % Positive C. briggsae none N/A N/A C. remanei none N/A N/A C. brenneri none N/A N/A Percentage glutamine, asparagine and proline residues in full length and C-terminal regions of mut-16 orthologs mut-16 (full length) Q/N Q N P C. elegans (1-1050) 14.8% 7.4% 7.4% 10.1% C. briggsae (1-1038) 13.1% 7.0% 6.1% 12.0% C. remanei (1-1104) 15.0% 8.3% 6.7% 10.0% C. brenneri (1-779) 10.4% 5.0% 5.4% 7.7% C. japonica (1-1022) 12.5% 6.4% 6.1% 13.4% mut-16 (C-term.) Q/N Q N P C. elegans ( ) 28.5% 18.2% 10.3% 17.0% C. briggsae ( ) 21.6% 14.4% 7.2% 24.4% C. remanei ( ) 27.6% 19.1% 8.5% 17.0% C. brenneri ( ) 11.8% 7.5% 4.3% 15.6% C. japonica ( ) 23.6% 13.3% 10.3% 24.3% Phillips et al. S-9
10 Table S4. sirna reads from high sirna yielding transposons*. Sequence Name wt RPM mut-16 RPM mut-16/wt Chromosome Genomic Cooridinates Transposon Classification Y102A5D V Retrotransposon-like T27C V Retrotransposon-like T05C V Unknown T05H V DDE Transposase family T23B V Unknown Y20F I Retrotransposon-like K03D IV Unknown F14D II Tc8/TURMOIL1/Tourist K03D IV Retrotransposon-like T23B V Retrotransposon-like F58H IV Retrotransposon-like R06A II DDE Transposase family Y73B3A X DDE Transposase family R07H IV Tc5 C03A V Retrotransposon-like Y43F4A III Retrotransposon-like R13D V Retrotransposon-like Y48G1BM I Retrotransposon-like Y37H2A V Retrotransposon-like F30F I Tc4 H25K IV Retrotransposon-like F07B V Retrotransposon-like K10E I Tc4 Y38H6C V Tc4 C44B IV Retrotransposon-like F21E X LINE2D_CE K06C V Retrotransposon-like Y54C5B II Tc5 M IV Tc5 Y38F2AR IV Tc5 F57G V TURMOIL2/Tourist none II Tc5 none IV Tc5 F26H I DDE Transposase family T07G IV Tc5 T25G X Unknown F38E X LINE2A_CE ZK II Retrotransposon-like C40A II Retrotransposon-like C27H IV DDE Transposase family H28G X DDE Transposase family Y51A2D V DDE Transposase family W06G V DDE Transposase family T14G X DDE Transposase family K03H IV DDE Transposase family C52D IV DDE Transposase family ZK V Retrotransposon-like W04G I DDE Transposase family T16A II Unknown Multiple* Multiple ~32 loci Tc1 family Multiple* Multiple ~14 loci rte-1 family Multiple* Multiple ~22 loci Tc3 family * Tc1, rte-1 and Tc3 family transposons produced too few sirna reads to be classified as high sirna yielding transpsosons after accounting for copy number. Phillips et al. S-10
11 Table S5. Strains used in this study. Strain name Genotype Description N2 wild-type NL1810 mut-16(pk710) I - outcrossed 4x NL3531 rde-2(pk1657) I - outcrossed 4x TW332 mut-2(r459) I - outcrossed 4x WM30 mut-2(ne298) I - outcrossed 4x NL1820 mut-7(pk720) III - outcrossed 4x GR1747 mut-15(tm1358) V - outcrossed 4x NL1838 mut-14(pk738) V - outcrossed 4x WM187 avr-14(ad1302) rrf-1(nec1) ego-1(om97)/ht2[qis48] I; +/ht2 III; avr-15(ad1051) glc-1(pk54) V GR1748 unc-119(ed3) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV mut-16::gfp GR1749 mut-7(pk720) unc-119(ed3) III; mgsi13[mut-7p::mut-7::gfp::mut-7-3'utr] IV mut-7::gfp GR1750 mgsi14[mut-7p::mut-7::mcherry::mut-7-3'utr] II; mut-7(pk720) unc-119(ed3/ed9) III mut-7::mcherry GR1751 unc-119(ed3/ed9) III; mgsi15[mut-14p::mut-14::mcherry::mut-14-3'utr] II; mut-14(pk738) V mut-14::mcherry GR1752 mut-2(ne298) I; unc-119(ed3) III; mgsi16[mut-2p::mut-2::gfp::mut-2-3'utr] IV mut-2::gfp GR1753 mut-2(ne298) I; unc-119(ed3/ed9) III; mgsi17[mut-2p::mut-2::mcherry::mut-2-3'utr] II mut-2::mcherry GR1754 unc-119(ed3/ed9) III; mgsi18[mut-15p::mut-15::mcherry::mut-15-3'utr] II; mut-15(tm1358) V mut-15::mcherry GR1755 rde-2(pk1657) I; unc-119(ed3) III; mgsi19[rde-2p::rde-2::gfp::rde-2-3'utr] IV rde-2::gfp GR1756 rde-2(pk1657) I; unc-119(ed3/ed9) III; mgsi20[rde-2p::rde-2::mcherry::rde-2-3'utr] II rde-2::mcherry GR1757 unc-119(ed3) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV; [nmy-2::pgl-1::mrfp; unc-119] mut-16::gfp; pgl-1::rfp GR1759 GR1760 GR1761 GR1762 GR1763 GR1764 GR1765 GR1766 GR1767 GR1768 GR1769 GR1770 GR1771 GR1772 GR1773 GR1774 GR1775 GR1776 GR1777 GR1778 GR1779 GR1780 GR1781 GR1782 GR1783 mut-2(ne298) I; unc-119(ed3) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV mut-7(pk720) unc-119(ed3) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV rde-2(pk1657) I; unc-119(ed3) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV unc-119(ed3) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV; mut-14(pk738) V unc-119(ed3) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV; mut-15(tm1358) V mut-2(ne298) I; mut-7(pk720) unc-119(ed3) III; mgsi13[mut-7p::mut-7::gfp::mut-7-3'utr] IV rde-2(pk1657) I; mut-7(pk720) unc-119(ed3) III; mgsi13[mut-7p::mut-7::gfp::mut-7-3'utr] IV mut-7(pk720) unc-119(ed3) III; mgsi13[mut-7p::mut-7::gfp::mut-7-3'utr] IV; mut-14(pk738) V mut-7(pk720) unc-119(ed3) III; mgsi13[mut-7p::mut-7::gfp::mut-7-3'utr] IV; mut-15(tm1358) V mut-16(pk710) I; mut-7(pk720) unc-119(ed3) III; mgsi13[mut-7p::mut-7::gfp::mut-7-3'utr] IV mut-2(ne298) I; mut-7(pk720) unc-119(ed3) III; mgsi16[mut-2p::mut-2::gfp::mut-2-3'utr] IV mut-2(ne298) rde-2(pk1657) I; unc-119(ed3) III; mgsi16[mut-2p::mut-2::gfp::mut-2-3'utr] IV mut-2(ne298) I; unc-119(ed3) III; mgsi16[mut-2p::mut-2::gfp::mut-2-3'utr] IV; mut-14(pk738) V mut-2(ne298) I; unc-119(ed3) III; mgsi16[mut-2p::mut-2::gfp::mut-2-3'utr] IV; mut-15(tm1358) V mut-2(ne298) mut-16(pk710) I; unc-119(ed3) III; mgsi16[mut-2p::mut-2::gfp::mut-2-3'utr] IV mut-2(ne298) rde-2(pk1657) I; unc-119(ed3) III; mgsi19[rde-2p::rde-2::gfp::rde-2-3'utr] IV rde-2(pk1657) I; mut-7(pk720) unc-119(ed3) III; mgsi19[rde-2p::rde-2::gfp::rde-2-3'utr] IV rde-2(pk1657) I; unc-119(ed3) III; mgsi19[rde-2p::rde-2::gfp::rde-2-3'utr] IV; mut-14(pk738) V rde-2(pk1657) I; unc-119(ed3) III; mgsi19[rde-2p::rde-2::gfp::rde-2-3'utr] IV; mut-15(tm1358) V rde-2(pk1657) mut-16(pk710) I; unc-119(ed3) III; mgsi19[rde-2p::rde-2::gfp::rde-2-3'utr] IV mut-2(ne298) I; mgsi18[mut-15p::mut-15::mcherry::mut-15-3'utr] II; unc-119(ed3/ed9) III; mut-15(tm1358) V mgsi18[mut-15p::mut-15::mcherry::mut-15-3'utr] II; mut-7(pk720) unc-119(ed3/ed9) III; mut-15(tm1358) V rde-2(pk1657) I; mgsi18[mut-15p::mut-15::mcherry::mut-15-3'utr] II; unc-119(ed3/ed9) III; mut-15(tm1358) V mgsi18[mut-15p::mut-15::mcherry::mut-15-3'utr] II; unc-119(ed3/ed9) III; mut-14(pk738) mut-15(tm1358) V mut-16(pk710) I; mgsi18[mut-15p::mut-15::mcherry::mut-15-3'utr] II; unc-119(ed3/ed9) III; mut-15(tm1358) V GR1784 mut-2(ne298) I; mgsi15[mut-14p::mut-14::mcherry::mut-14-3'utr] II; unc-119(ed3/ed9) III; mut-14(pk738) V GR1785 mgsi15[mut-14p::mut-14::mcherry::mut-14-3'utr] II; mut-7(pk720) unc-119(ed3/ed9) III; mut-14(pk738) V GR1786 rde-2(pk1657) I; mgsi15[mut-14p::mut-14::mcherry::mut-14-3'utr] II; unc-119(ed3/ed9) III; mut-14(pk738) V GR1787 mgsi15[mut-14p::mut-14::mcherry::mut-14-3'utr] II; unc-119(ed3/ed9) III; mut-14(pk738) mut-15(tm1358) V GR1788 mut-16(pk710) I; mgsi15[mut-14p::mut-14::mcherry::mut-14-3'utr] II; unc-119(ed3/ed9) III; mut-14(pk738) V GR1789 GR1790 GR1791 GR1792 avr-14(ad1302) ego-1(om97) rrf-1(nec1)/ht2[qis48] I; mut-7(pk720) unc-119(ed3)/ht2 III; mgsi13[mut-7p::mut-7::gfp::mut-7-3'utr] IV avr-14(ad1302) ego-1(om97) rrf-1(nec1)/ht2[qis48] I; unc-119(ed3)/ht2 III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV drh-3(tm1217) I/hT2[qIs48] I; unc-119(ed3)/ht2 III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV ekl-1(ok1197) I/hT2[qIs48] I; unc-119(ed3)/ht2 III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV GR1793 mut-2(ne298) I; mgsi17[mut-2p::mut-2::mcherry::mut-2-3'utr] II; unc-119(ed3/ed9) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV mut-2::mcherry; mut-16:gfp GR1794 mgsi18[mut-15p::mut-15::mcherry::mut-15-3'utr] II; unc-119(ed3/ed9) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV; mut-15(tm1358) V mut-15::mcherry; mut-16::gfp GR1795 rde-2(pk1657) I; mgsi20[rde-2p::rde-2::mcherry::rde-2-3'utr] II; unc-119(ed3/ed9) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV rde-2::mcherry; mut-16::gfp GR1796 mgsi15[mut-14p::mut-14::mcherry::mut-14-3'utr] II; unc-119(ed3/ed9) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV; mut-14(pk738) V mut-14::mcherry; mut-16::gfp GR1797 mgsi14[mut-7p::mut-7::mcherry::mut-7-3'utr] II; mut-7(pk720) unc-119(ed3) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3'utr] IV mut-7::mcherry; mut-16::gfp GR1798 mut-2(ne298) I; mgsi14[mut-7p::mut-7::mcherry::mut-7-3'utr] II; mut-7(pk720) unc-119(ed3) III; mgsi16[mut-2p::mut-2::gfp::mut-2-3'utr] IV mut-7::mcherry; mut-2::gfp GR1912 ego-1(om97) rrf-1(nec1) I; mgsi24[ego-1p::3xha::ego-1::3xflag::rrf-1::rrf-1 3 UTR] II; unc-119(ed9) III HA::ego-1 FLAG:rrf-1 GR1913 ego-1(om97) rrf-1(nec1) I; mgsi24[ego-1p::3xha::ego-1::3xflag::rrf-1::rrf-1 3 UTR] II; unc-119(ed9) III; mgsi2[mut-16p::mut-16::gfp::mut-16-3 UTR] IV HA::ego-1 FLAG:rrf-1; mut- 16::gfp Phillips et al. S-11
12 Table S6. Primers used in this work. Cloning primers mut-16 promoter mut-16 gene mut-16 3'UTR mut-7 promoter mut-7 gene mut-7 3'UTR mut-2 promoter mut-2 gene mut-2 3'UTR rde-2 promoter rde-2 gene rde-2 3'UTR mut-15 promoter mut-15 gene mut-15 3'UTR mut-14 promoter mut-14 gene mut-14 3'UTR ego-1 promoter ego-1 gene/region between ego-1 and rrf-1 region between ego-1 and rrf-1 rrf-1 gene/3'utr ctgtgtttttctgccgtcgac tttctgcaaatatcgagtatttgag atgtccgaaagtgatgatgattatc gtttcggatatcatctttcaaaacg gaatttacttgttcctattactgttc aatacgtcatatcataaattcacaaag aacaataatggagaacgtttgtcg tttcgaaacggacaagcttgattc atggaagaagaaccgtacaaaag acattcctggctggtgctc attcgaaatcccgttccccttc gtagagccttgcgaaatgaac aaacactaatacatttcacttacaatc tgttgttactacaaaaaatattcaattattattc atgtctcaaccaaataaagatcag tacaaatgagttccgacagtag tcttaatatgtcgatgttttgtc ggattgtagttagtttgattgc gcaatgtttaaatatacggtaac ctgaatagcaagaaaatgaatatag atgcataatgggtaccattcttattttc ctttcgaactgaaataataatttgaatag aaactgcacaatcgacaaatcac gtttctctttgtaaacctgaaaaatg caaaaatcgattatttaacaaaaacg ttctttgatacttatctaaatacttc atggataactccaatggaatcg aacaggaacagctgatgttgc atatgtatttttcttccaacttctc acttcccgtctattaatctgtaaaac cgtcgattttgcagagttttgc ctgaaaaatatgattttctcgatc atgtcatatccctggagtaac agcccatgagtcgatattgttg aataattgatttactgtcagtagtatttag tgattcccattcctttgtttttg atggctgaagctgtttgctg tgttgcgaggattcgggata ggggacgaaggttatcgtgg ccgacatgacgaggagtctg agaggacacatcaacatctag gacgaggagtctgaaaaattttat tcggaacgaagtcatggattc aatgttcttggatcgaagacc Phillips et al. S-12
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