SRX20276910 GSM7316359_r1 SRP436840 PRJNA971123 SRS17604164 GSM7316359 GSM7316359 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276911 GSM7316368_r1 SRP436840 PRJNA971123 SRS17604174 GSM7316368 GSM7316368 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276912 GSM7316369_r1 SRP436840 PRJNA971123 SRS17604165 GSM7316369 GSM7316369 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276913 GSM7316370_r1 SRP436840 PRJNA971123 SRS17604173 GSM7316370 GSM7316370 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276914 GSM7316371_r1 SRP436840 PRJNA971123 SRS17604166 GSM7316371 GSM7316371 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276915 GSM7316372_r1 SRP436840 PRJNA971123 SRS17604177 GSM7316372 GSM7316372 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276916 GSM7316373_r1 SRP436840 PRJNA971123 SRS17604171 GSM7316373 GSM7316373 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276917 GSM7316374_r1 SRP436840 PRJNA971123 SRS17604175 GSM7316374 GSM7316374 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276918 GSM7316375_r1 SRP436840 PRJNA971123 SRS17604169 GSM7316375 GSM7316375 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276919 GSM7316384_r1 SRP436840 PRJNA971123 SRS17604183 GSM7316384 GSM7316384 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276920 GSM7316385_r1 SRP436840 PRJNA971123 SRS17604184 GSM7316385 GSM7316385 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276921 GSM7316386_r1 SRP436840 PRJNA971123 SRS17604180 GSM7316386 GSM7316386 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276922 GSM7316387_r1 SRP436840 PRJNA971123 SRS17604185 GSM7316387 GSM7316387 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276923 GSM7316388_r1 SRP436840 PRJNA971123 SRS17604192 GSM7316388 GSM7316388 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276924 GSM7316389_r1 SRP436840 PRJNA971123 SRS17604182 GSM7316389 GSM7316389 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276925 GSM7316390_r1 SRP436840 PRJNA971123 SRS17604167 GSM7316390 GSM7316390 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276926 GSM7316391_r1 SRP436840 PRJNA971123 SRS17604168 GSM7316391 GSM7316391 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276927 GSM7316360_r1 SRP436840 PRJNA971123 SRS17604170 GSM7316360 GSM7316360 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276928 GSM7316361_r1 SRP436840 PRJNA971123 SRS17604172 GSM7316361 GSM7316361 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276929 GSM7316362_r1 SRP436840 PRJNA971123 SRS17604176 GSM7316362 GSM7316362 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276930 GSM7316363_r1 SRP436840 PRJNA971123 SRS17604178 GSM7316363 GSM7316363 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276931 GSM7316364_r1 SRP436840 PRJNA971123 SRS17604179 GSM7316364 GSM7316364 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276932 GSM7316365_r1 SRP436840 PRJNA971123 SRS17604181 GSM7316365 GSM7316365 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276933 GSM7316366_r1 SRP436840 PRJNA971123 SRS17604186 GSM7316366 GSM7316366 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276934 GSM7316367_r1 SRP436840 PRJNA971123 SRS17604193 GSM7316367 GSM7316367 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276935 GSM7316376_r1 SRP436840 PRJNA971123 SRS17604187 GSM7316376 GSM7316376 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276936 GSM7316377_r1 SRP436840 PRJNA971123 SRS17604196 GSM7316377 GSM7316377 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276937 GSM7316378_r1 SRP436840 PRJNA971123 SRS17604191 GSM7316378 GSM7316378 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276938 GSM7316379_r1 SRP436840 PRJNA971123 SRS17604188 GSM7316379 GSM7316379 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276939 GSM7316380_r1 SRP436840 PRJNA971123 SRS17604195 GSM7316380 GSM7316380 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276940 GSM7316381_r1 SRP436840 PRJNA971123 SRS17604189 GSM7316381 GSM7316381 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276941 GSM7316382_r1 SRP436840 PRJNA971123 SRS17604190 GSM7316382 GSM7316382 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276942 GSM7316383_r1 SRP436840 PRJNA971123 SRS17604198 GSM7316383 GSM7316383 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276943 GSM7316392_r1 SRP436840 PRJNA971123 SRS17604197 GSM7316392 GSM7316392 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276944 GSM7316393_r1 SRP436840 PRJNA971123 SRS17604194 GSM7316393 GSM7316393 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000 SRX20276945 GSM7316394_r1 SRP436840 PRJNA971123 SRS17604199 GSM7316394 GSM7316394 ChIP-Seq GENOMIC ChIP For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing. Illumina NovaSeq 6000