of NOBOX. Supplemental Figure S1F shows the staining profile of LHX8 antibody (Abcam, ab41519), which is a

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1 SUPPLEMENTAL MATERIALS AND METHODS LHX8 Staining During the course of this work, it came to our attention that the NOBOX antibody used in this study had been discontinued. There are a number of other nuclear marks specific to growing oocytes that could be used in place of NOBOX. Supplemental Figure S1F shows the staining profile of LHX8 antibody (Abcam, ab41519), which is a suitable alternative marker for p10 oocyte isolation. Staining was done as with NOBOX, at a 1:500 dilution of primary antibody. KDM1A/KDM1B RNA-Seq For RNA-Seq analysis of KDM1A KO, KDM1B KO, and their corresponding control oocytes, three biological replicates were prepared for each genotype. Each sample, which contained 100 GV oocytes collected from 2-3 mice, was lysed directly in 1 μl of prelude direct lysis buffer (Nugen). RNA was then subject to amplification using the ovation RNA-seq system v2 (Nugen). Amplified cdna was fragmented using Covaris and paired-end libraries were then constructed using the TruSeq RNA Sample Preparation Kit v2 (Illumina) starting from end repair. The libraries were sequenced using a 2x76 bases paired end protocol on the Illumina HiSeq 2000 instrument. Each pair of reads represents a cdna fragment from the library. KDM1A/KDM1B Genome-Wide DMR Analysis For the unbiased DMR analysis, a 15-CpG sliding window was used to define DMRs between KDM1A- and KDM1Bdeficient samples and their relevant controls. Chi-squared tests were done using total methylated and unmethylated calls within each window, comparing KDM1A- and KDM1B-deficient samples to their relevant controls. Overlapping differentially methylated windows that met a p <0.01 threshold were merged to generate the final differentially methylated regions (DMRs). These DMRs were included in downstream analyses if they had at least 5 CpGs covered by at least 1 read in each control and knockout replicate.

2 SUPPLEMENTAL FIGURES AND LEGENDS Supplemental Figure S1. FACS Strategies for Primary and Growing Oocyte Isolation. (A) Immunofluorescence of presort and postsort E18.5 ovarian cells sorted using SYCP3, stained with Mouse Vasa Homolog (MVH) antibody, SYCP3 antibody and DAPI. (B) Immunofluorescence of presort and postsort p10 ovarian nuclei sorted using DNA content and NOBOX, stained with SYCP3 antibody, NOBOX antibody and DAPI. (C) Imagestream analysis shows that nuclei isolation at p10 gives clean, undamaged NOBOX+ nuclei. (D) Full FACS gating strategy for E18.5 primary oocyte isolation. (E) Full FACS gating strategy for p10 growing oocyte nuclei isolation. (F) FACS analysis of E18.5 (left panel) and p10 (center and right panels) ovaries stained with DAPI, SYCP3 and LHX8. Shown in all graphs is the 4N population as marked by DAPI staining.

3 Stewart271353_FigS1 A Postsort SYCP3 Presort MVH C DAPI Brightfield B DAPI SYCP3 NOBOX Presort D E F Postsort Nobox Merge DAPI

4 Supplemental Figure S2. Supplemental to Figure 2. 2i mouse embryonic stem cell K4me3 signal (logtransformed corrected read count) at 4kb centered on CGIs, separated into GVmeth and GVunmeth CGIs and by CpG density. Black line, median; dashed lines, 1.5x interquartile range. Data from Marks and colleagues (Marks et al. 2012). Reference Marks H, Kalkan T, Menafra R, Denissov S, Jones K, Hofemeister H, Nichols J, Kranz A, Stewart AF, Smith A, et al The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149:

5 Stewart271353_FigS Corrected Read Count < >7 CpG Density per 100 bp GVmeth GVunmeth

6 Supplemental Figure S3. Supplemental to Figure 3. (A) Trend plot showing p10 K36me3 from -2kb to +5kb from TSS at genes, separated by expression level. (B) Boxplots showing p10 K36me3 enrichment (log-transformed corrected read count) at genes containing GVmeth and GVunmeth CGIs, parsed by expression level. Black line, median; dashed lines, 1.5x interquartile range. (C) E18.5 and p10 K36me3 signal (log-transformed corrected read count) at 4kb centered on CGIs, separated into GVmeth and GVunmeth CGIs and by CpG density. Black line, median; dashed lines, 1.5x interquartile range. (D) Pie charts showing the proportion of TSS, intragenic and intergenic CGIs as annotated by RNA-Seq that fall into protective, permissive and unenriched states at E18.5 and p10.

7 Stewart271353_FigS3 A 1.2 p10 K36me3 B 4 n.s. n.s. p10 K36me3 n.s. n.s. n.s % 60-80% 40-60% 20-40% 0-20% Corrected Read Count TSS % 20-40% 40-60% 60-80% % Expression Level Genes containing GVmeth CGI Genes containing GVunmeth CGI C K36me3 D 8 E18.5 p10 Corrected Read Count Corrected Read Count < >7 E18.5 p10 TSS Intragenic Intergenic < >7 CpG Density per 100 bp GVmeth GVunmeth Protective State Permissive State Unenriched

8 Supplemental Figure S4. Supplemental to Figure 4. (A) Piecharts showing the proportion of DMRs within KDM1A- and KDM1B-knockout oocytes that are hypermethylated and hypomethylated relative to a flox/flox, ZP3-Cre- (KDM1A) or wildtype (KDM1B) control. (B) Stacked barchart showing the proportion of KDM1A and KDM1B hyper- and hypodmrs that overlap promoters, gene bodies, and intergenic regions with respect to the canonical oocyte transcriptome. (C) Heatmap showing DNA methylation levels in KDM1A-deficient, KDM1B-deficient and the corresponding wild-type oocytes of CGIs who met coverage requirements and had >75% methylation in at least one sample (799). (D) Scatterplots comparing RNA- Seq (log-transformed corrected read count) in KDM1A- and KDM1B-deficient oocytes with controls. Overlaid are genes that overlap a hyperdmr (red dots) and genes that overlap a hypodmr (blue dots) as determined using unbiased sliding-window analysis. (E) Boxplots showing K36me3, K4me2 and K4me3 E18.5 and p10 enrichment (log-transformed corrected read count) at KDM1B HypoCGIs that either do or do not transition from protective to permissive states between E18.5 and p10. Black line, median; dashed lines, 1.5x interquartile range. (F) Genome screenshot showing DNA methylation over the Gata4 locus in normal and KDM1A- or KDM1B-deficient mature oocytes. Gata4 shows loss of methylation along the entire transcription unit in KDM1A-null oocytes, and more targeted loss of methylation at an intragenic CGI in KDM1B-null oocytes. Shown is a 100 bp window with a 100 bp step.

9 Stewart271353_FigS4 A DMRs in KDM1A KO Hypermethylated 30.7% (n=3425) DMRs in KDM1B KO Hypermethylated 0.22% (n=85) B Promoter Gene Body Intergenic 20 Hypomethylated 69.3% (n=7899) Hypomethylated 99.78% (n=45,102) Hyper Hypo Hyper Hypo KDM1A KDM1B C D % Methylation KDM1A KDM1B KDM1B KO KDM1A KO CTRL KDM1B WT KDM1A CTRL WT KO KO overlapping HyperDMR overlapping HypoDMR E KDM1B HypoCGIs F K36me3 K4me2 K4me3 Corrected Read Count E18.5 p10 E18.5 p10 E18.5 p10 mrna CGI KDM1A KDM1B KO CTRL KO WT E18.5 Protective, p10 Permissive No Transition E18.5 Protective, p10 Permissive No Transition E18.5 Protective, p10 Permissive No Transition chr14:

10 SUPPLEMENTAL TABLES Table S1. CGI Annotations at E18.5 and p10; separate Excel file. Table S2. Annotations of CGIs at e18.5. Table S3. Annotations of CGIs at p10. Table S4. Annotations of GVmeth CGIs that Transition from Protective to Permissive States. Table S5. Overlap between KDM1A and KDM1B Hyper- and HypoCGIs. Table S6. Methylation at Imprinted gdmrs in KDM1A- and KDM1B-Deficient Oocytes; separate Excel file. Table S7. E18.5 and p10 ChIP-Seq Library Information; separate Excel file. Table S8. KDM1A/KDM1B PBAT Library Information; separate Excel file.

11 Table S2. Annotations of CGIs at E ' most TSS alttss Intragenic Intergenic Total GVmeth 230 (11.2%) 103 (5.0%) 1003 (49.0%) 711 (34.8%) 2047 Gvunmeth 9023 (46.2%)355 (1.8%) 1680 (8.6%) 8489 (43.4%) Table S3. Annotations of CGIs at p10. 5' most TSS alttss Intragenic Intergenic GVmeth 140 (6.8%) 158 (7.7%) 1551 (75.8%) 198 (9.7%) 2047 Gvunmeth 9126 (46.7%)1383 (7.1%) 2863 (14.6%) 6175 (31.6%) Table S4. Annotations of GVmeth CGIs that Transition from Protective to Permissive States. p10 5' most TSS Intragenic Intergenic alttss E18.5 n ' most TSS Intragenic Intergenic alttss Table S5. Overlap between KDM1A/KDM1B Hyper- and HypoCGIs. KDM1A HyperCGI HypoCGI KDM1B n HyperCGI HypoCGI