Figure S1. Replication initiation sites at efficient ORIs do not coincide with nucleosomedepleted

Transcription

1 Figure S1. Replication initiation sites at efficient ORIs do not coincide with nucleosomedepleted regions in the mouse genome. Typical examples of SNS profiles (Cayrou et al., 11) and nucleosome occupancies (Teif et al., 12) at CGI-ORIs, shown as custom tracks on the UCSC genome browser. The scale bar corresponds to 5 kb. Figure S2. High-resolution nucleosome analysis at ORIs co-localising with active promoters. (A) Maps of the active promoter-ori regions analysed and NuSA on mononucleosomal DNA isolated after incubating nuclei with increasing concentrations of MNase. All symbols are like in Figure 1C. If a nucleosome is well positioned (ie, it occupies the same position in all cells), a primer pair located totally within this nucleosome will yield a high enrichment relative to that on undigested DNA. On the contrary, if one primer is within and the other primer is outside of the nucleosome, the apparent enrichment will be low. Thus, a positioned nucleosome array will generate peaks and valleys, with the valleys corresponding to linker regions between nucleosomes (Sekinger et al., 05). (B) NuSA on α-h3 immunoprecipitated DNA derived from crosslinked chromatin and MNase digestion. ChIP experiments were performed at low salt (upper panels) or high salt (lower panels) conditions accounting for different nucleosome stabilities in different salt concentrations, and qpcr data represents nucleosomal DNA enrichments relative to untreated DNA. (C) NuSA on mononucleosomal DNA derived from crosslinked chromatin and low MNase treatment at Vps45 ORI region (full MNase titration is shown in Fig. 2C). (D) Same genomic maps as in A showing the nucleosome positioning and occupancies inferred from the data. Nucleosomes were numbered according to their location relative to the TSS. Light grey and red ovals represent stable and labile nucleosomes, respectively. Figure S3. High-resolution nucleosome analysis at ORIs co-localising with bivalent CGIpromoters. (A) Maps of the bivalent promoter-ori regions analysed and NuSA on 1

2 mononucleosomal DNA isolated after incubating nuclei with increasing concentrations of MNase. Symbols are as in Figure 2. Only 281 bp could be analysed at the Atp2b3 promoter due to the presence of repetitive DNA at both sides of the TSS (hatched lines). (B) NuSA on α-h3 immunoprecipitated DNA derived from crosslinked chromatin and MNase digestion. ChIP experiments were performed at low salt (upper panels) or high salt (lower panels) conditions. (C) Same genomic maps as in A showing the nucleosome positioning and occupancies inferred from the data. Figure S4. High-resolution nucleosome analysis at ORIs mapping at non-promoter regions. Same analysis as in Figure S2 and S3 at the ORIs located within the transcribed regions of the genes Pias3, Syde1 and Scl7a14. In these cases, ChIP assays were only performed at low salt conditions. Figure S5. Comparison of high-resolution nucleosome maps and genome-wide data. Alignment of nucleosome maps of promoter-oris generated by NuSA on mononuclosomes obtained by combining various experimental procedures (A) with those from the genomewide analysis of Teif et al., 12 (B). Figure S6. Effect of emetine in the amount and length of replication intermediates. (A) Southern analysis of SNS derived from identical number of asynchronous ES cells growing in normal conditions (- emetine) or after 1h exposure to 2 µm emetine (+ emetine) (Burhans et al., 1991). Lanes 1 to 3 correspond to SNS purified from three consecutive sucrose gradient fractions (-0, and 0-10 nt, respectively) treated twice with λ-exonuclease, separated by alkaline agarose electrophoresis, blotted, and visualized by hybridization with labelled total mouse genomic DNA. (B) SNS enrichment at Mecp2 ORI region measured by high-resolution qpcr scanning on the replication intermediates preparations shown in (A) 2

3 derived from either non-treated (left panels, blue lines) or emetine-treated (right panels, red lines) cells. Data were normalized to the values obtained at a negative region in each fraction. Maps above the graphs represent the Mecp2 ORI nucleosomal array derived from the data shown in Figure S2. The horizontal blue line represents the identified ORI region in Sequeira- Mendes et al., 09. Figure S7. High-resolution analysis of short replication intermediates abundance and nucleosome configuration at ORIs not associated with gene promoters. Same analysis as in Figure 3 at the ORIs located within the transcribed regions of the genes Pias3, Syde1 and Scl7a14. Full NuSa analysis is shown in Figure S4 and replication initiation profiles relative to a non-ori region are shown in Figure S9. Figure S8. High-resolution analysis of short replication intermediates abundance at promoter-oris. Maps with the nucleosome patterns and histograms of SNS abundance at the highly efficient ORIs co-localising within the promoter regions of Mecp2 and Atp3b3 (A) and at the slightly less efficient ORIs co-localising within the promoter regions of Vps45, Haus7 and Hs6st2. SNS enrichments were normalised to those obtained at a non-ori region in each sucrose gradient fraction. Figure S9. High-resolution analysis of short replication intermediates abundance at low efficient ORIs. Same analysis as in Figure S8 at the ORI regions located at the gene bodies of the genes Pias3, Syde1 and Slc7a14. Figure S10. Overrepresented short DNA fragments derived from nucleosome-free regions at LaminB2 ORI in human 293T cells and at a LaminB2 transgene in mouse ES cells. 3

4 qpcr measurements of amplicon abundance at the LaminB2 ORI on total genomic DNA or -0 nt SNS derived from human 293T cells (A-C) or mouse ES cells carrying the laminb2 transgene (D-F). All data were normalised to the foremost 3 primer pair. Due to its overrepresentation on genomic DNA preparations, short DNA fragments are unveiled only by qpcr when primer set efficiencies are normalized using the cloned version of a locus instead of genomic DNA to construct the standard curves (A, B, D, E) (Gómez and Antequera, 08). Solid red bars highlight the amplicons that are overrepresented on total genomic DNA and therefore, their abundance is higher than one (A, D). When similar measurements using cloned DNA to build the standard curves were applied to short SNS (B, E), the overrepresented fragments become more apparent as by their small size, they are enriched in the -0 nt SNS preparation. As a consequence, the replication initiation profile obtained in the smallest SNS fraction shows and extra peak at that precise position when cloned DNA is employed to build the standard curves (compare panels B and C or E and F). This effect, however, does not occur in longer SNS preparations (data not shown), confirming that DNA synthesis does not elongate from the sites from which such short overreplicated fragments derived (Gómez and Antequera, 08). Solid grey bars highlight the amplicons that identify true initiation sites from which replication elongates bidirectionally, as demonstrated when measuring SNS abundance on preparation of replication intermediates of increasing sizes and using sonicated gdna to build the standard curves (C, F) and Figure 5. As expected, the relative abundance of bona fide initiation sites is higher when SNS are normalised with cloned DNA to build the standard curves (B, E) than when normalised with gdna preparations as they also contain replication intermediates (C, F). It should be noted that, although interrogating the same genomic region, the primer pairs employed in panels A, B, D and E are different to those used in panels C, F and in Figure 5. This is due to our strict requirements of amplification efficiencies for each primer pair, which are different when using cloned standards or genomic standards in the qpcr reactions (see Methods). Primer 4

5 sequences used in reactions with cloned DNA to build the standard curves are available upon request. Figure S11. Schematics of the LaminB2 ORI and TIMM13 mrna levels in human and mouse cells. (A) Upper panel, genomic region on human chromosome 19 showing the endogenous location of the 2.2 kb LaminB2 ORI fragment used in the knock-in experiments. Positions are from ncbi Build Lower panel, detailed scheme of the transgenic construct. The fragment contains the start site of bi-directional DNA replication mapped at singlenucleotide resolution (Abdurashidova et al., 00) represented by two diverging blue arrows emanating from the extended in vivo-protected region that coincides with the zone where Orc2, Orc1 and Cdc6 locate during the G1 phase of the cell cycle (Abdurashidova et al., 00); the region positive for Orc2 and Orc1 binding in ChIP experiments (Ladenburger et al., 02); and the 1.2 kb NruI/BglII fragment functioning as a replicator at ectopic positions in the human genome (Paixao et al., 04). The position of the CGI and the region analysed by NuSA are also shown. Black arrows indicate the location and directionality of the major TSS (Carninci et al., 06). (B) RT-PCR analysis of TIMM13 and Hprt in human 293T cells (h), mouse ES cells (m) and mouse ES cells carrying the LaminB2 transgene (t). The expected RT-PCR product is marked by a white arrow. cdna synthesis conditions and Hprt primer pairs are as in Fig. 4. TIMM13 primers used in the RT-PCR reactions were TIMM 41 and TIMM 42 (Table S1). Genomic DNA derived from 293T (h) and mouse ES (m) cells was also included as a control in the PCR reactions. M, 1kb plus DNA ladder. Table SI. Primer sets and conditions used in the NuSA and SNS abundance quantifications. 5

6 SNS (Cayrou et al., 11) Nucleosomes (Teif et al., 12) Figure S1-Lombraña et al

7 A Mecp2 Haus7 Vps45 Nucleosomal DNA enrichment MNase B 2 Nucleosomal DNA enrichment High salt Low salt D C Nucleosomal enrichment Figure S2-Lombraña et al

8 A Atp2b3 Hs6st2 Nucleosomal DNA enrichment MNase B Nucleosomal DNA enrichment Low salt High salt C Figure S3-Lombraña et al

9 A Pias3 Slc7a14 Syde1 Nucleosomal DNA enrichment MNase B Nucleosomal enrichment C Figure S4-Lombraña et al

10 A Haus7 Vps45 Atp2b3 Mecp2 High MNase Low MNase Teif et al., 12 H3-ChIP B Figure S5-Lombraña et al

11 A m emetine B SNS enrichment relative to negative region emetine + emetine nt nt 0-10 nt Figure S6-Lombraña et al

12 A Pias3 Slc7a14 Syde1 Nucleosome enrichment B Nucleosome enrichment C D SNS enrichment relative to proximal flank nt -0 nt 0-10 nt Figure S7-Lombraña et al

13 A Mecp2 Atp2b SNS enrichment relative to the negative rehion nt -0 nt 0-10 nt B Vps45 Haus7 Hs6st SNS enrichment relative to the negative rehion nt -0 nt 0-10 nt Figure S8-Lombraña et al

14 Pias3 Syde1 Slc7a14 SNS enrichment relative to negative region nt -0 nt 0-10 nt Figure S9-Lombraña et al

15 A hlaminb2 D LaminB2 transgene ORC Relative enrichment gdna vs clone B E ORC Relative enrichment SNS vs clone C F ORC 5 Relative enrichment SNS vs gdna Figure S10-Lombraña et al

16 LMNB2 2429K 2426K 2427K TIMM K 2424K 2422K Chr K TMPRSS9 2428K A CGI loxp BglII OPR ORC loxp NruI 826 bp analysed by NuSA B t + - m + - h + - m h M TIMM13 0 Hprt Figure S11-Lombraña et al

17 Primers and amplicons used in the NuSA experiments Region Primer Sequence 1 Anneal. Mecp2 (572 bp) Atp2b3 (281 bp) Haus7 (589 bp) Mecp 1 Mecp 2 Mecp 3 Mecp 4 Mecp 5 Mecp 6 Mecp 7 Mecp 8 Mecp 9 Mecp 10 Mecp 11 Mecp 12 Mecp 13 Mecp 14 Mecp 15 Mecp 16 Mecp 17 Mecp 18 Mecp 19 Mecp Mecp 21 Mecp 22 Mecp 23 Mecp 24 Mecp 25 Mecp 26 Mecp 27 Mecp 28 Atp 1 Atp 2 Atp 3 Atp 4 Atp 5 Atp 6 Atp 7 Atp 8 Atp 9 Atp 10 Atp 11 Atp 12 Haus 1 Haus 2 Haus 3 Haus 4 Haus 5 Haus 6 Haus 7 Haus 8 Haus 9 Haus 10 Haus 11 Haus 12 Haus 13 Haus 14 GAGGGCGTCATTCGAAGGTG AATCGCGAGCGACGGTTCTC GCTGAGAACCGTCGCTCGC CGCCGGTGGTGGCTTTCTC GAGAAAGCCACCACCGGCG CGACACGGCTGGCGGATG CATCCGCCAGCCGTGTCG CTTGCTGGGGGGCGGGTAG CTACCCGCCCCCCAGCAAG GTGAGTGGGACCGCCAAGG CCTTGGCGGTCCCACTCAC GCCGAGCGGAGGAGGAGG CTCCTCCGCTCGGCGCG GCTGTGGTAAAACCCGTCCGG TTTTCCGGACGGGTTTTACC CGCTCCCTCCTCTCGGAG CTCCGAGAGGAGGGAGCG GACGTCTGCCGTGCGGGGT ACCCCGCACGGCAGACGTC GTGCAGCAGCACACAGGCTG GACCAGCCTGTGTGCTGCTG CTCGACAAAGAGCAAGGGGTG CACCCCTTGCTCTTTGTCGAG CCAGCCTGGGCTCCACAAC GTTGTGGAGCCCAGGCTGG CAATTGAGGGCGTCACCGCT AGCGGTGACGCCCTCAATTG CCTCTTTTCCCTGCCTAAAC TCCCTCCGCGCTCAGAGCTG GACGCTGCGCGCTCGACTC GAGTCGAGCGCGCAGCGTC AGGGCTGTGGCCGAGTGCTC TGGGGAGCACTCGGCCACAG AGCAGTGCGGAGCAGGACCC GGGTCCTGCTCCGCACTGCT CCAAGGACGGAAAGGCCGGG TCCGTCCTTGGAGCTGATCG GCTGTGGCTTTCGCTGTTGC CGCAACAGCGAAAGCCACAGC GCACAACTTACCCCGGCATG AAGCTTTGCCAGGGCCTG GCCATTTGGAGCTTCCTGTG TCCACAGGAAGCTCCAAATG TCCTTGATAGGGGACCAGG TGGTCCCCTATCAAGGAG GAAGCTGAAGGTACTGTGAG CTGGCTCACAGTACCTTCAG GTGATGACTGCGTGGTCAAG CTTGACCACGCAGTCATCAC TGCCGGGTCTGATGACAG TAGTAGCTGTCATCAGACCC GCGTGAACCAGTTGTAGTTC CTGAACTACAACTGGTTCACG GGTGAGCTTTTAGCAGTGTG T re Amplicon Size 64ºC 59 bp 66ºC 56 bp 66ºC 71 bp ºC 75 bp 64ºC 53 bp 66ºC 58 bp ºC 76 bp 64ºC 48 bp 62ºC 56 bp 68ºC 57 bp 66ºC bp 66ºC 54 bp 66ºC 58ºC 56 bp 66ºC 45 bp 66ºC 79 bp 66ºC 57 bp 64ºC 58 bp 66ºC 64 bp 66ºC 64 bp 64ºC 70 bp 58ºC 58 bp 56ºC ºC 64 bp 62ºC 55 bp 56ºC 76 bp 56ºC 65 bp

18 Hs6st2 (514 bp) Vps45 (541 bp) Haus 15 Haus 16 Haus 17 Haus 18 Haus 19 Haus Haus 21 Haus 22 Haus 23 Haus 24 Haus 25 Haus 26 Haus 27 Haus 28 Hst 1 Hst 2 Hst 3 Hst 4 Hst 5 Hst 6 Hst 7 Hst 8 Hst 9 Hst 10 Hst 11 Hst 12 Hst 13 Hst 14 Hst 15 Hst 16 Hst 17 Hst 18 Hst 19 Hst Hst 21 Hst 22 Hst 23 Hst 24 Hst 25 Hst 26 Hst 27 Hst 28 Vps 1 Vps 2 Vps 3 Vps 4 Vps 5 Vps 6 Vps 7 Vps 8 Vps 9 Vps 10 Vps 11 Vps 12 Vps 13 Vps 14 Vps 15 Vps 16 CGCCGCCACACTGCTAAAAG GATGGTTGGCTTCGCTCCTG GAGCGAAGCCAACCATCG GAAAAGAGGGTGGGCCTTTG CGTCACAAAGGCCCACCCTC ACAGAGCGGCAGCCCAATGG TTGGGCTGCCGCTCTGTC CTTCTCTGCACCCTGCTCCC GGGAGCAGGGTGCAGAGAAG GCCTTTAGCCCGCCTCTG ACAGAGGCGGGCTAAAGGC CCTCCTCCTCTCTGCCTCTC AGGCAGAGAGGAGGAGGC CTGCCTCTAAAAGGAGCTACTC TCTGGATGTGCAGGAACACG CGATCTCCTGCGCAAGGTAG CTACCTTGCGCAGGAGATCG TGAGAGTTCGGCCAGGTTTG ACAAACCTGGCCGAACTCTC GTTCAGCTCCCCGGTGCCG CGGCACCGGGGAGCTGAAC CACAGAATGCCAGCTCCTCC GGAGGAGCTGGCATTCTGTG GGTGATCGTCCTCCAATACG CGTATTGGAGGACGATCACC ACAAGCTGCTGCTGGCTTTC CAAAGCCAGCAGCAGCTTGT CAGCGTCGGGAACATGGATG ATCCATGTTCCCGACGCTGG GCGGGATTGACGTAACGAGC GCTCGTTACGTCAATCCCGC AGCGGGAGCGAGGCGAGC GCTCGCCTCGCTCCCGCA GGAAAGGGGCGCAACGGTGG CCACCGTTGCGCCCCTTTCC GCCGCCGTGGTGGAGTACT AGTACTCCACCACGGCGGC GAAGGAAGGAAGCGGGGAAG CTTCCCCGCTTCCTTCCTTC TGTGCGGCAGAAGCCACAGC GCTGTGGCTTCTGCCGCACA AACGGCCAGCGGGATGGAG AGTTAGAGCTGGTTAGACTG CTGGGAAAGTTGTAGAATGC TCTACAACTTTCCCAGTGCC CTGATGACGATGATCAAGTC GACTTGATCATCGTCATCAG GTTCCGCTGTAGAACTTAGC AATGCTAAGTTCTACAGCGG ATAAAGGAAGCTCTCCCTTC TAAAGGAAGGGAGAGCTTCC TCATGGATAAAGAAACGGTGAG CCGTTTCTTTATCCATGAGAAG AAATGATAGAGGACAGCGGG CGCTGTCCTCTATCATTTTG TCAATTCGCCACCATGAATG ATTCATGGTGGCGAATTGAC TAATTCAGCCAGGAAAGTGG 64ºC 82 bp 64ºC 66ºC 57 bp 66ºC 57 bp 64ºC 58 bp 62ºC 51 bp 58ºC 67 bp 64ºC 68 bp 64ºC 65 bp 66ºC bp 66ºC 49 bp 64ºC 64ºC bp 64ºC 49 bp 66ºC 51 bp 64ºC 66ºC 57 bp 66ºC 55 bp 64ºC 48 bp 64ºC 54 bp 66ºC 55 bp 56ºC 56 bp 56ºC 45 bp 56ºC 46 bp 56ºC 62 bp 58ºC 67 bp 58ºC 57 bp ºC 68 bp ºC 63 bp

19 Pias3 (8 bp) Syde1 (512 bp) Vps 17 Vps 18 Vps 19 Vps Vps 21 Vps 22 Vps 23 Vps 24 Vps 25 Vps 26 Pias 1 Pias 2 Pias 3 Pias 4 Pias 5 Pias 6 Pias 7 Pias 8 Pias 9 Pias 10 Pias 11 Pias 12 Pias 13 Pias 14 Pias 15 Pias 16 Pias 17 Pias 18 Pias 19 Pias Pias 21 Pias 22 Pias 23 Pias 24 Pias 25 Pias 26 Pias 27 Pias 28 Syde 1 Syde 2 Syde 3 Syde 4 Syde 5 Syde 6 Syde 7 Syde 8 Syde 9 Syde 10 Syde 11 Syde 12 Syde 13 Syde 14 Syde 15 Syde 16 Syde 17 Syde 18 Syde 19 Syde CCACTTTCCTGGCTGAATTAACC TTTGGCGACCGGAAGCAG TTCCGGTCGCCAAAGCCTC CCCAGTATCGGAGCTACCCG CGGGTAGCTCCGATACTGG TAGCTGCTGAGTCTGAGTCCC TCAGACTCAGCAGCTAAGCG TTCCACTCCCTACCGAGAAG TTCTCGGTAGGGAGTGGAAG TACTCAGGACCAGAAGCCAG TATGGGCTGGATGGTGAGTG AAGGGCCAGCCATGACTTAG TGAGCTAAGTCATGGCTGGC GAGACCTGTGGGTGGTTAAG CTTAACCACCCACAGGTCTC CCCTCTTCTTACTCTCTGGC TCAGCCAGAGAGTAAGAAGAG CTGATGAGCTTTCGATGGTC GACCATCGAAAGCTCATCAG TGACAGGGCAGTGCTTCTTG AAGAAGCACTGCCCTGTCAC TCATACCCTTTGCTTCCAGG AGCCCTTCCTGGAAGCAAAG GGCAGTTCTGACTTCTGTCC GGGACAGAAGTCAGAACTGCC TCTTCCCTGTCCCAAAGCTC CGAGCTTTGGGACAGGGAAG GAGCTAGCCAAGCGCAGATG CTGCGCTTGGCTAGCTCTC AGGTCAGGGCTCTGTGAGTG CACTCACAGAGCCCTGACCTC TGCCCAGTGTGCCCATTG AATGGGCACACTGGGCAG AGGTGGGTACTCATGTAGCGG GCTACATGAGTACCCACCTG GGGTCAGTGTTCTACCTTGG GCTGACATCCAAGGTAGAAC AAACAGGTCTGGAGAGGGTG TCTTAACTCCCCCTGGCTAC TGTCAAACTAGCTGGCAGG CCCTGCCAGCTAGTTTGACA GGAGTGAGAAAGATGATTGG CCAATCATCTTTCTCACTCC TTAGCCCTCATGGAGCTCAG CTGAGCTCCATGAGGGCTA ACCCACGGATCATCTGTCC AAGGACAGATGATCCGTGGG CACTCCCTCCCCAGCATCA TGATGCTGGGGAGGGAGTG CTCCTTGGAACCCCTTAAAC GTTTAAGGGGTTCCAAGGAG GTGAGCTGGCTGCCCCCAC GTGGGGGCAGCCAGCTCAC GAAAGTGTGTGGGGCAGATC GATCTGCCCCACACACTTTC CAGTCCCCAGGCCAGGTGC GCACCTGGCCTGGGGACTG CTGCAGCTTCTGGTAGAACG 64ºC 61 bp 64ºC 53 bp ºC 65 bp ºC 58 bp ºC 69 bp ºC 61 bp 58ºC bp 56ºC 69 bp 56ºC 59 bp ºC 62 bp ºC 61 bp 58ºC 70 bp 62ºC 56 bp 64ºC 63 bp 62ºC 53 bp 64ºC 74 bp 62ºC 58 bp 56ºC 62 bp 56ºC 76 bp 62ºC 56 bp 58ºC 51 bp 58ºC 45 bp 62ºC 55 bp 64ºC 42 bp ºC 41 bp 62ºC 62 bp 64ºC 57 bp 64ºC 51 bp 64ºC 43 bp

20 Slc7a14 (582 bp) TIMM13 (826 bp) Syde 21 Syde 22 Syde 23 Syde 24 Syde 25 Syde 26 Syde 27 Syde 28 Syde 29 Syde 30 Slc 1 Slc 2 Slc 3 Slc 4 Slc 5 Slc 6 Slc 7 Slc 8 Slc 9 Slc 10 Slc 11 Slc 12 Slc 13 Slc 14 Slc 15 Slc 16 Slc 17 Slc 18 Slc 19 Slc Slc 21 Slc 22 Slc 23 Slc 24 Slc 25 Slc 26 TIMM13 1 TIMM13 2 TIMM13 3 TIMM13 4 TIMM13 5 TIMM13 4 TIMM13 6 TIMM13 7 TIMM13 8 TIMM13 9 TIMM13 10 TIMM13 11 TIMM13 12 TIMM13 13 TIMM13 14 TIMM13 15 TIMM13 16 TIMM13 17 TIMM13 18 TIMM13 19 TIMM13 TIMM13 21 CGTTCTACCAGAAGCTGCAG AGCAGAGCCCCGTGTTTTC GAAAACACGGGGCTCTGCT GCTAACATTGTCGGAACAGC GCTGTTCCGACAATGTTAGC ACAACTGGCCGTTCGACTTG CAAGTCGAACGGCCAGTTGT GCCTCAGTCCTCCTTGCATC AGATGCAAGGAGGACTGAGG GATGAGGGGAGTCAGGACAC AGGTTATGCCTGGGGATG AGGTGGAAACTTCTTCCAACTC TGAGAGTTGGAAGAAGTTTC GGTTTCTTAGTACATCAGCC GGCTGATGTACTAAGAAACC CTCTCCGGAGTAAGACTAAC GCGCGGTTAGTCTTACTCCG AACAGAACTCGGAGGCCCTG ATCAGGGCCTCCGAGTTCTG GGACGAGTAGCAAAGCGAAAAG TTCGCTTTGCTACTCGTCCG CCGAGGACAAAGGCTTCTATTAC TAGAAGCCTTTGTCCTCGGC GTTCTCCTACGCCACCGAG CTCGGTGGCGTAGGAGAAC GCTACGATGTGGATGATCCC CATCCACATCGTAGCGCTGG AATCAGCGCCCGAGAGCAAG CTCTCGGGCGCTGATTTC CCCTCCCAACAGGTATGCTC AGCATACCTGTTGGGAGGGG CGGGCCCCAAGATAGCTG CAGCTATCTTGGGGCCCG ACCCCAGCCCTAGTCAGGTG AGATCACACCTGACTAGGGC CAAGTCTTTCCAAAATGTGC GAGGCTTTAGCCGCGACGTC GCTAACATTGTCGGAACAGC CCAGGGGTGGCCCTGTC GGTGAAAGTGCAGATCGCCG CTCCATTATGAGCCCTGGGTC GGCCTCGGTCCGGTTGACT CCCGAAATCGGAGCCG GGCCTCGGTCCGGTTGACT ACCGAGGCCGCGTGCG GGACTCCGTTTCCCGTGGT ACCACGGGAAACGGAGTCC GCGCGGTCGTGTGGGA TAGCTCGTGTAGGTAACGGC GGGCCATTCAAGGTCGCGCG GCTCATGCGGAGGCCTGG CAACCACGGGTAGCTCGTGTAGG CCTACACGAGCTACCCGTGGTTG CAGGGCCTCCCTCTTCCCG CGGGAAGAGGGAGGCCCTG GTAGCCCCCCTACTCCCCGG CCGGGGAGTAGGGGGGCTAC GAGTACAAAGTGATCGGCCTCGG 62ºC 48 bp 62ºC 55 bp 62ºC 65 bp 66ºC 58 bp 62ºC 62 bp 58ºC 72 bp 56ºC 56 bp 56ºC 62 bp 64ºC 63 bp 64ºC 58 bp 62ºC 73 bp 62ºC 69 bp 62ºC 56 bp 66ºC 59 bp 62ºC 74 bp 64ºC 56 bp 64ºC 57 bp 58ºC 66 bp 64ºC * 119 bp 64ºC * 95 bp 64ºC * bp 64ºC * 58 bp 66ºC * 76 bp 66ºC * 61 bp 62ºC 72 bp 64ºC * 51 bp 64ºC * 49 bp 64ºC * 44 bp 64ºC * 51 bp

21 TIMM13 22 TIMM13 23 TIMM13 24 TIMM13 25 TIMM13 26 TIMM13 27 TIMM13 28 TIMM13 29 TIMM13 30 TIMM13 31 TIMM13 32 TIMM13 33 TIMM13 34 TIMM13 33 TIMM13 35 TIMM13 36 CCGAGGCCGATCACTTTGTACTC GGTGCCTCGTGCGCATG TGCCTCCAGCTCGTCCCG CAACACGCTGTATAGACGCGCC GGCGCGTCTATACAGCGTGTTG GATGCGACCGGGCTCCG CGGAGCCCGGTCGCATC TGGGACCCTGCCCTTTTTTTTC TCAGCTTGTGCAACAGCGTC GCTAGTGTAAACAGGACCCAGGCG GAGGGATCTTTCTTAGACATCC GGTTCTGCCTCTGAGTTTATTCC GAGGCCCCGGCTCGAG GGTTCTGCCTCTGAGTTTATTCC GCTGTGACGCTCGCTGG CTGAGGGACTCCTCAGTCC 64ºC * 77 bp 64ºC * 71 bp 66ºC * 47 bp 68ºC * 83 bp 66ºC * 82 bp 64ºC 84 bp 64ºC 48 bp 62ºC 74 bp Amplifications were run for cycles in a final volume of µl with 1 µl of input DNA, 2 µl of 10x Qiagen HotStar Taq buffer, 1.2 µl of 25 mm MgCl 2, 0.4 µl of 10 mm dntps, 0.5 µl of SYBR Green, 0.5 U of Taq polymerase, and 1.5 µl of primers (5 µm each). 1- Sequence is given in the 5 to 3 direction * These amplicons were generated using the following qpcr cycles: 1 minute at 94ºC 30 seconds at the annealing temperature and 30 seconds at 72ºC.

22 Additional primers and amplicons used in the SNS-scan experiments Region Primer Sequence Mecp2 Mecp5' F TTGTGGCGCACTCTCCCAAC Mecp5' R CACACAGACTGGCGCGCGTG Mecp3' F GCATCCAATGCTCTTTGTGC Mecp3' R GTCTCTTGTTGAGCATTTGT Atp2b3 Atp5' F ACTTGATAACCTTTAGGGATCAG Atp5' R CAGATACATTTCCCCAGAGG Atp F GCACGCGCATCTGTGTCTAG Atp R CTATGCCTGCCTGGATCCCC Atp3' F CACATGTTCCTACACTGTCC Atp3' R ACTGTCCATGCTAGCGTCAG Haus7 Haus5' F AAACTGCAAGGAAAAACTCC Haus5' R TTTCTTAGTCCATCCTGAGG Haus3' F TATTTCTATCCTCCACAAGG Haus3' R GAAAACAAGCAACCAAAAGC Hs6st2 Hst5' F GTAGTGTCCAGTCCTTGCGG Hst5' R TGAGTGAGTGTGGGGAAAGC Hst3' F CTTTTCATCTGGGAAAACAG Hst3' R ATTAGCTAGGAAATGTCTGC Vps45 Vps5' F TACAGGACGAGAATTGGAAC Vps5' R GACCACTGGGATTAACGGAA Vps3' F AAGGGAATGAATACAAGGAG Vps3' R CCAGGGAGCTTTAGGAAC Pias3 Pias5 'F ATTATTGATGGGTAGGGCTC Pias5' R GTAAGCCATAAAGAGGGGAG Pias3 'F TCATCTCTCCAGCACTACGG Pias3' R GGTGTCCTGTTCATCTAGCG Syde1 Syde5' F CTTCCAGGAAGCCTTCTCAC Syde5' R AGAAGCAGTAAGGAGGGGAG Syde3' F GATAGCGGCTGATGACAGAG Syde3' R TAAGAAGTACTCGCACAGGC Slc7a14 Slc5' F CATGTCTGCCAATGCTTTTC Slc5' R ACCCACCATCAGAGCTTGAG Slc3' F AAGGCAGAATGCTCGTACTC Slc3' R AAAACCTGACCACCACTTCC TIMM13 TIMM 3 F AAAGGCCTGAAGGAGGTGCCAC TIMM 3 R CAGCCAGTGCAGCTTGGATCTC TIMM13 37 CTCGAGCCGGGGCCTCTG TIMM13 38 TAGCTACACTAGCCAGTGACCTTTTTCC TIMM13 39 TGAAAGTCCATGCCAGCC TIMM13 CAACCTGCAAAAACGGAGC Non-ORI Neg F CCAAACTTCAGCAGTTTTCC region Neg R GAAATTGTCTTGCAAGAGGA Anneal. T re Amplicon Size 66ºC 55 bp ºC 43 bp 58ºC 74 bp 64ºC 89 bp 58ºC 71 bp 56ºC 89 bp 53ºC 84 bp 62ºC 70 bp 58ºC 99 bp ºC 101 bp 56ºC 74 bp 56ºC bp ºC 57 bp 62ºC 92 bp 58ºC 69 bp ºC 88 bp ºC 57 bp 64ºC 68 bp 64ºC 88 bp 64ºC 58ºC 61 bp