C. elegans strains and culture conditions

All strains were cultured on NGM (Nematode Growth Medium) agar plates at 20 °C, unless otherwise noted, using standard methods. CF512 and SS1167 were maintained at 15 °C, and HBR1280 and HBR1281 were maintained at 24 °C. The following strains were used: N2 (Bristol) as wild type, CB1489 him-8(e1489) IV, CF512 rrf-3(b26) II; fem-1(hc17ts) IV, DH245 fem-2(b245ts) III, SS818 mes-3(bn35) I/hT2-GFP (I;III), SS1167 mes-3(bn35) I/hT2-GFP (I;III); fem-2(b245ts) III, HBR1280 oxTi75[eft-3p::GFP::H2B::tbb-2 3′UTR + unc-18( + )], HBR1281 oxTi411[eft-3p::TdTomato::H2B::unc-54 3′UTR + Cbr-unc-119( + )] III; ddIs32[yfp::gpr-1(synthetic, CAI 1.0, artificial introns)].

Isolation of C. elegans males and purification of sperm

The following protocols were partially adapted from refs. 33,38. These protocols isolate highly enriched populations of spermatids, which have compacted nuclei that have completed meiotic division but have not developed motility structures to form motile spermatozoa. As these cells have undergone sperm-specific DNA compaction, we refer to these spermatids as mature sperm or “sperm” for short throughout the text. Mature sperm from C. elegans were collected and purified from him-8(e1489) males. A mutation in him-8 causes X chromosome non-disjunction during meiosis and thus generates a high percentage (~ 30%) of males39. Large cultures of him-8(e1489) worms (5–10 million) were synchronously grown from starved L1s in fernbach flasks containing S medium and HB101 Escherichia coli, shaking at 270 r.p.m. at 20 °C for ~ 62 h (MaxQ 3000 Benchtop shaker, Barnstead Lab-Line). The mixture of gravid hermaphrodites and adult males (day 1 adults) was collected using a separatory funnel. An equal volume of worm suspension and 60% ice-cold sucrose was mixed in a 25 mL tube and centrifuged for 5 min at 1000 × g. Worms contained in the top-layer were collected on 20-micron Nytex mesh (CellMicroSieves, BioDesign, Inc.) and rinsed with S-basal, after which males were allowed to wiggle through the mesh for at least for 30 min; gravid hermaphrodites remained on top of the mesh. The males were transferred to a 15-micron mesh, rinsed with S-basal several times to remove any larvae and small debris, and collected from the top of the mesh. Filtering of males was repeated twice to yield 10–15 mL packed males of > 98% purity as determined under a dissecting microscope. Males were resuspended in 20 mL freshly prepared Squash Medium (50 mM HEPES, 1 mM MgSO 4 , 70 mM choline chloride, 5 mM CaCl 2 , 1 mM phenylmethylsulfonyl fluoride, 1 mg/mL bovine serum albumin, 190 mOsm, pH 6.5), centrifuged for 5 min at 1000 × g and settled on ice, and excess Squash Medium was removed. The packed males were pressed 2 mL at a time using a custom-made “Sperminator” apparatus: males were evenly placed on one Plexiglass “squash plate”, a second “squash plate” was placed on top, and the two plates were pressed gently together using the “Sperminator” at 1000 psi for 10 s to squeeze sperm out of male worms, while preserving intact worms to prevent contamination from ruptured worm tissues. The collected sperm were filtered through 20-, 15-, 10-, and two rounds of 5-micron Nytex mesh to eliminate contamination by non-sperm cells and spermatocytes. Purified sperm were collected in a 1.5 mL eppendorf tube by centrifugation (1700 × g for 5 min), rinsed with Squash Medium, and again filtered through a 5-micron mesh. Sperm quantity (20–100 million sperm) was quantified using a hemocytometer, and sperm purity (~ 99%) was estimated by microscopic analysis (DIC imaging and DNA staining). Pelleted sperm were flash frozen in liquid nitrogen and kept at −80 °C.

Preparation of sperm chromatin for ChIP

Frozen sperm (10~50 million) were thawed on ice, rinsed once in 1 mL chilled Micrococcal Nuclease (MNase) Buffer (50 mM HEPES pH 7.5, 110 mM NaCl, 40 mM KCl, 2 mM MgCl 2 , 1 mM CaCl 2 ), and fixed in 1 mL freshly prepared Fix Solution (1% formaldehyde in MNase Buffer) for 52 s, while constantly pipetting up and down. After adding glycine (final 125 mM) to stop cross-linking, sperm were rinsed once with 1 mL chilled MNase Buffer. Fixed sperm were treated with Lysis Buffer (final 0.2% NP-40 and 0.5% sodium deoxycholate) for 5 min on ice and digested with MNase for 5 min at 37 °C at a density of 0.1 million sperm per μL (5 units of MNase per 1 million sperm). To each tube, EDTA (final 10 mM), protease inhibitor (Roche, #04693159001, final 1 × after a tablet was resuspended in water), and Triton X-100 (final 1%) were added, and each tube was nutated at 4 °C for at least 2 h to facilitate chromatin solubilization. The digested sperm chromatin was mixed with two volumes of 150 mM salt FA Buffer (50 mM of HEPES pH 7.5, 1 mM EDTA pH 7.4, 1% Triton X-100, 0.1% sodium deoxylcholate, 150 mM NaCl) and treated with a Covaris S2 model (duty cycle 20%, intensity 8, cycle per burst 200, 60 s per cycle, total 5 cycles) to liberate sperm chromatin from the insoluble material. The entire content was transferred to a 1.5 mL siliconized tube, and centrifuged at 16,000 × g for 15 min at 4 °C. The supernatant, which contains fragmented and soluble sperm chromatin, was transferred to a new siliconized tube, flash frozen in liquid nitrogen, and stored at − 80 °C. Agarose gel electrophoresis (1.2% stained with SYBR Green) and an Agilent 2100 Bioanalyzer System were used to confirm that the majority of soluble chromatin was in mononucleosomes.

ChIP-seq from sperm

Soluble sperm chromatin from ~ 3.3 million sperm was thawed on ice, brought up to 110 μL with 150 mM salt FA Buffer supplemented with sarkosyl (final 1%). As “input” 10 μL was saved. To the remaining 100 μL soluble sperm chromatin, 1 μg antibody was added, and ChIP was performed using an IP-Star Compact Automated System (Diagenode) according to the manufacturers’ instructions with the following settings and reagents: “indirect method” with 100 μL reaction, 80 μL DynabeadsTM (M-280, sheep anti-mouse IgG, Life Technologies); IP for 12 h, bead incubation for 2 h; 10 min washes at middle speed with 1 M salt FA Buffer, 500 mM salt FA Buffer, TEL Buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8), and TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA); elution in Elution Buffer (1% SDS and 250 mM NaCl in 1X TE). Both IP and “input” were brought up to 300 μL with Elution Buffer with 1.5 μL 20 mg/mL proteinase K and incubated for 2 h at 55 °C and then overnight at 65 °C to reverse crosslinks. The next day, the magnetic beads were removed from the ChIP samples using a magnetic stand and sperm DNA was extracted using phenol–chloroform extraction, using the Phase Lock Gel system (5 PRIME #2302810). Sperm DNA was precipitated overnight at − 80 °C in ethanol with glycogen as carrier, and the resulting DNA pellets were resuspended in 15 μL nuclease-free water. One third of the DNA (5 μL) was used for ChIP-quantitative PCR (qPCR) to check ChIP efficiency, and libraries were prepared from the remaining 10 μL ChIP and “input” DNA using MicroPlex Library Preparation kit v2 (Diagenode, #C05010012). A real-time qPCR machine (Roche LightCycler 480) was used to monitor library amplification to avoid overamplification. Size selection was performed after library amplification using AMPure XP beads (Beckman Coulter, A63881) to enrich for 100–300 bp inserts. The final libraries were evaluated using an Agilent 2100 Bioanalyzer System (High Sensitivity DNA Analysis kit) and Quant-iT assay (Invitrogen, high-sensitivity double-stranded DNA). The multiplexed libraries were sequenced on either HiSeq4000 or HiSeq2500 rapid platforms at the Vincent J. Coates Genomics Sequencing Laboratory at University of California, Berkeley.

Isolation of C. elegans oocytes

The following protocols were partially adapted from refs. 40,41,42. C. elegans oocytes were collected and purified from hermaphrodites feminized by temperature-sensitive mutations in fem-1 and rrf-3 (previously known as fer-15) in strain CF51243. At the restrictive temperature, mutant hermaphrodites fail to make sperm and accumulate unfertilized oocytes in the uterus. Synchronized L1 larvae from CF512 were plated on 40–60 High Growth (HG) plates (2% peptone, 51 mM NaCl, 25 mM potassium phosphate, 5 μg/mL cholesterol, 1 mM CaCl 2 , 1 mM MgCl 2 , 2.5% agar) seeded with E. coli OP50 and grown for ~ 55 h at 15 °C until worms reached late L3 stage. Worms were then upshifted to 25 °C for 24–36 h to feminize them. Visual inspection under a dissecting microscope confirmed that day 1 feminized adults contained only unfertilized oocytes, and that no fertilized embryos or L1 larvae were present on the plates. Approximately 1 million feminized adults were washed from the HG plates with Egg Salts Buffer (25 mM HEPES pH 7.4, 118 mM NaCl, 48 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 ), and washed 3 × in a 50 mL conical tube to remove excess E. coli and other debris. After washing, worms were pelleted by centrifugation, excess buffer was removed, and densely packed worms were transferred to a 15 cm petri dish on ice. Worms were chopped with a clean razor blade for 5 min, until extruded gonads and liberated oocytes were visible under a dissecting microscope. Oocytes and carcass fragments were poured over a 45-micron mesh (NTX45, Dynamic Aqua-Supply LTD). Oocytes passed through the mesh, while carcass fragments remained above the mesh. The flow-through was filtered through a 20-micron mesh to collect oocytes above the mesh, while smaller debris went through the mesh. An aliquot of unfixed oocytes was saved for 4′,6-diamidino-2-phenylindole (DAPI) staining and RNA isolation to estimate the quality and purity, measured as the number of oocytes/total cells in multiple fields of view under a dissecting microscope. The purity of the first and second replicates was > 95% and > 90%, respectively. Oocytes were fixed in 2.2% formaldehyde in Egg Salts Buffer for 5 min, quenched in 125 mM glycine to stop cross-linking, and then washed twice in M9 Buffer (3 g KH 2 PO 4 , 6 g Na 2 HPO 4 , 5 g NaCl, H 2 O to 1 L). Fixed oocytes were pelleted, flash frozen in liquid nitrogen, and stored at − 80 °C.

ChIP-seq from oocytes

Fixed oocytes (0.5–1 million) were thawed on ice and resuspended in chilled MNase Buffer. Oocytes were sonicated for 5 cycles with a tip sonicator (5 rounds of 5 s on and 25 s off). Following sonication, oocytes were pre-warmed to 25 °C for 5 min, followed by addition of 250 units MNase. A pilot digestion time-course experiment was performed to identify the optimal digestion time, which yielded the majority of chromatin as mononucleosomes, as measured by Agilent bioanalyzer analysis. As a result of the pilot experiment, oocyte chromatin was digested with MNase for 50 min. Digestion was halted by addition of EDTA (final 10 mM) and incubated on ice for 5 min. Protease inhibitor (Roche, #04693159001, final 1 × in water), Triton X-100 (final 1%), and sodium deoxycholate (final 0.1%) were added to the digested oocyte chromatin. The oocyte chromatin extract was split into separate aliquots, and ChIPed in parallel with different antibodies. Dynabeads were washed 3 × with cold FA Buffer. Each antibody (1 μg) was nutated with beads at 4 °C for ~ 2 h. After saving 40 μL oocyte extract as “input,” oocyte extract was added to the beads, supplemented with sarkosyl (1% final), and nutated overnight at 4 °C. The next day, the beads were washed twice in FA Buffer, once in 1 M salt FA Buffer, 500 mM salt FA Buffer, and TEL Buffer, and then twice in TE Buffer. Immunocomplexes were eluted from the beads by incubation with Elution Buffer at 65 °C for 15 min with gentle vortexing. RNA was digested with RNase A for 1 h at room temperature, followed by proteinase K treatment for 2 h at 55 °C. Crosslinks were reversed by incubation at 65 °C overnight, followed by phenol–chloroform extraction, and DNA precipitation in ethanol with linear acrylamide at − 80 °C for 4 h. ChIPed oocyte DNA was resuspended in 15 μL nuclease-free water. Oocyte libraries were prepared with the NEBNext Ultra DNA library Prep Kit (NEB) following the manufacturers’ instructions. AMPure beads were used for size selection after amplification to enrich for fragments corresponding to mononucleosomes. Libraries were sequenced on an Illumina HiSeq2500 platform at the Princeton High Throughput Sequencing Facility.

ChIP-seq from early embryos

Data are from ref. 19.

Antibodies used for ChIP

Mouse monoclonal antibodies for H3K36me3 (HK00001, now marketed as Wako MABI0333, #300–95289), H3K27me3 (HK00013, now marketed as Wako MABI0323, #309–95259), and H3K4me3 (Wako MABI0304, #305–34819) are described in ref. 44 and were validated as in ref. 45.

Analysis of ChIP-seq data

Raw sequence reads from the Illumina HiSeq (50 bp single-end reads) were mapped to the C. elegans genome (Wormbase version WS220) using Bowtie with default settings46. MACS2 was used to call peaks and to create bedgraph files with the following settings: -g ce --bdg --keep-dup = auto --broad --broad-cutoff = 0.01 --nomodel --extsize = 25047. Bedgraph files were scaled to 10 million total autosomal reads (X chromosome reads were not included for scaling, due to the different X:A ratios between sperm, oocytes, and early embryos) and converted to bigwig using the bedGraphToBigWig UCSC Genome Browser tool48. These were displayed in the UCSC Genome Browser (visibility = full, autoScale = off, windowingFunction = Mean + whiskers, smoothingWindow = 2). To display gene-level ChIP signal in scatter plots (e.g., Fig. 2c), the mean read coverage for each protein-coding gene was calculated using the bigWigAverageOverBed UCSC Genome Browser tool48 using WS220 gene start and end annotations. For H3K4me3, a histone mark typically found in promoters, the read coverage was calculated from 500 bp upstream of the gene start annotation to 500 bp downstream. These gene read coverages were log 2 transformed after a pseudo-count of 1 was added. To account for slightly varying background noise levels in the different ChIP-seq samples, the gene read coverages were z-scored based on the average and standard deviation of all autosomal gene read coverages. Specifically, the read coverages for all genes were centered by subtracting the average of the read coverages of autosomal genes and then divided by the standard deviation of the autosomal gene read coverages. One adaptation was made for H3K27me3: the 30th percentile of autosomal gene read coverage, instead of the average, was chosen as the baseline signal and was subtracted from all gene read coverages genome-wide. This is because H3K27me3 occupies ~ 2/3 of the C. elegans genome and the autosomal average gene read coverage did not seem like an appropriate baseline to center the H3K27me3 ChIP signal. The Principal Component Analysis in Supplementary Fig. 4d was performed on these z-scored autosomal gene read coverages. The prcomp function in R was used. Metagene analysis was performed using the R package “SeqPlots” with default settings49.

MNase-seq

Sperm were fixed as described for sperm ChIP-seq, and 1–2 million sperm were digested with increasing amounts of MNase for 5 min at 37 °C. Early embryos were harvested from hermaphrodites that contained only a couple of embryos and fixed as described in ref. 15 . Early embryos (500–750 μL) were thawed on ice and resuspended in 2 mL of Dounce Buffer (0.35 M sucrose, 15 mM HEPES pH 7.5, 0.5 mM EGTA, 0.5 mM MgCl 2 , 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5% Triton X-100) supplemented with protease inhibitors (Roche, #04693159001) and dounce-homogenized ~ 10 times until the majority of embryos were disrupted. The homogenized embryos were spun at 380 × g for 1 min at 4 °C and the supernatant containing nuclei was transferred to a new tube. This was repeated one more time. The nuclei contained in the supernatant were collected by centrifugation at 4000 × g for 10 min at 4 °C and resuspended in MNase Buffer. Approximately 5 million embryo nuclei were digested with increasing amounts of MNase at 37 °C for 30 min. Digested chromatin from sperm and early embryos was reverse-crosslinked overnight at 65 °C in Elution Buffer supplemented with 1.5 μL 20 mg/mL proteinase K. DNA was extracted using phenol–chloroform. The degree of MNase digestion was assessed using agarose gel electrophoresis and an Agilent 2100 Bioanalyzer. Illumina sequencing libraries were constructed with DNA extracted from the mononucleosome-enriched digestions that were comparable between sperm and early embryos, using NEBNext Ultra DNA Library Prep Kit (NEB, E7370). During library construction, size selection was performed on adaptor-ligated DNA before amplification to remove fragments larger than 250 bp (40 μL of AMPure beads were used for First Bead Addition based on the manufacturers’ instructions), and PCR-amplified DNA was purified using 1:1 AMPure XP beads, which removed fragments smaller than 100 bp. The final libraries were evaluated using an Agilent 2100 Bioanalyzer System and Quant-iT assay, and were sequenced at the Vincent J. Coates Genomics Sequencing Laboratory at University of California, Berkeley (50 bp paired-end read sequencing).

Analysis of MNase-seq data

Paired-end MNase-seq reads were mapped to the C. elegans genome (Wormbase version WS220) using Bowtie with default settings46. After mapping, fragments smaller than 140 bp were filtered out using bamtools 2.5.150. Bedtools51 was used to scale the remaining mapped fragments to 10 million autosomal reads and to generate bedgraph files. As for ChIP-seq, the bedGraphToBigWig tool was used to convert the bedgraph files to bigwig format. Bigwig files were displayed in the UCSC genome browser (visibility = full, autoScale = off, windowingFunction = Mean, smoothingWindow = 2). To assess whether there are nucleosome-occupancy differences in certain genomic regions between sperm and early embryos, the bigWigAverageOverBed UCSC Genome Browser tool48 was used to calculate the average MNase fragment coverage of 150 bp windows tiled every 50 bp and covering the whole genome for all sperm and embryo samples. Windows (150 bp) that had less than 1 fragment covering them in all four sperm and early embryo samples were excluded from further analysis, as these 150 bp windows cover genomic regions of very low mappability with the sequencing data available. About 2.2% of the genome was excluded this way. Density plots were made in R to show the distribution of 150 bp window fragment coverage for the autosomes and for the X chromosome for all four samples of sperm and early embryo MNase-seq. The analysis was repeated for 500 bp windows tiled every 250 bp, 1 kb windows tiled every 500 bp, 2 kb windows tiled every 1 kb, and 5 kb windows tiled every 2.5 kb.

RNA sequencing

For male spermatogenic germlines, samples were from N2 males (Figs. 2,3), CB1489 him-8(e1489) males (Figs. 2,3,4), and SS818 mes-3 (bn35) homozygous M + Z − males from heterozygous parents (M for maternal product and Z for zygotic product) (Fig. 4). For sperm, samples were from CB1489 him-8(e1489) males and SS818 mes-3(bn35) M + Z − males (Fig. 4). For hermaphrodite oogenic germlines, samples were from N2 (Figs. 2,3), DH245 fem-2(b245) (Figs. 2,3), SS1167 mes-3(bn35) (M + P − Z − and M + P + Z − where P is for paternal H3K27me3 by MES-3); fem-2(b245) (Fig. 4), HBR1280 oxTi75[eft-3p::GFP::H2B::tbb-2 3′UTR + unc-18( + )] (Fig. 5, control), “red-head” F1 progeny (from HBR1281 crossed with HBR1280; genetically identical to HBR1280) (Fig. 5), and F2 offspring from “red-head” F1s (genetically identical to HBR1280) (Fig. 5). Both spermatogenic and oogenic germlines were dissected from day 1 adults as described in ref. 24; 20–100 distal gonad arms were cut at the late-pachytene gonad bend. Mature sperm were released from adult males by cutting the males with a 30-gauge needle and collected with a pulled glass Pasteur pipette coated with Sigmacote (SL2 Sigma). Total RNA was extracted in TRIzol reagent (Invitrogen), ribosomal RNA was depleted using an NEBNext rRNA Depletion kit (E6310), and libraries were prepared using an NEBNext Ultra RNA Library Prep Kit for Illumina (E7530). Libraries were sequenced at the Vincent J. Coates Genomics Sequencing Laboratory at University of California, Berkeley (50 bp single-end read sequencing). For each genotype, 2–4 biological replicates were obtained. Sequence reads were processed as described in ref. 52. Briefly, TopHat253 was used to align the RNA-seq reads to the C. elegans transcriptome (WS220) with default parameters. HTSeq54 was used to obtain read counts per transcript (HTseq counts). Differentially expressed genes were determined with DESeq255 in R using HTSeq counts and a FDR < 0.05 as the significance cutoff. RPKMs were calculated by dividing HTseq counts by exonic transcript length obtained from Wormbase and scaling the total read counts per sample to 1 million reads. If a gene had multiple transcript isoforms, the longest was chosen. For Fig. 3d and Supplementary Fig. 3a,c, RPKMs were log transformed after adding a pseudo-count of 1. Genes with RPKM > 15 were called ‘expressed’ based on the expression of defined spermatogenic and oogenic gene sets (see below) in spermatogenic and oogenic germlines.

Single-molecule fluorescence in situ hybridization

smFISH analysis was performed on day 1 adult male and hermaphrodite gonads dissected from him-8(e1489) worms as in ref. 56. Biosearch Technologies designed, synthesized, and labeled the Stellaris probes. The pie-1, par-6, and nos-2 probes were coupled to Quasar 670, the mex-3 probe was coupled to Cal Fluor Red 590, and the cpg-2 probe was coupled to Cal Fluor Red 610. Each probe was resuspended in 250 μL of TE Buffer pH 8.0 and then further diluted to 1:30 for hybridization. The microscope and its settings are as in ref. 52. Fig. 3 and Supplementary Fig. 6 contain montages generated by splicing together contiguous images acquired with identical settings. Male and hermaphrodite pairs used identical confocal settings, with the exposure optimized for visualizing the male gonads. All images were processed identically with ImageJ and Adobe Illustrator. For both oogenic and male germlines, 10–60 gonads were visually examined under the microscope and at least 3 germlines were imaged in 1–3 separate experiment(s).

Measurement of fertility/sterility

For Fig. 4, feminized heterozygous mes-3(bn35)/hT2-GFP; fem-2ts worms were obtained by shifting their mothers from 15 °C to 24 °C at the L4 larval stage and selecting GFP + offspring. Feminized worms were mated with either homozygous mes-3(bn35) M + Z −; fem-2ts males or heterozygous mes-3(bn35)/hT2-GFP; fem-2ts males raised at 15 °C, and the fertility/sterility of their offspring grown at 15 °C was visually scored under the microscope. We found that having fem-2ts and mes-3 heterozygous in the mother’s genotype is essential for paternal-effect sterility, and thus represents a sensitized genetic background. Unpaired unequal-variance one-tailed Student’s t-tests were performed to calculate the significance of % sterile worms between the genetically identical M + P − vs. M + P + offspring.

Measurement of brood size

For Fig. 5, strain maintenance and experiments with HBR1280 and HBR1281 were performed at 24 °C, because the transgenes in these strains are prone to gene silencing. Cross-progeny from the matings shown in the figure were scored using a fluorescence microscope to identify those that arose from normal vs. atypical segregation. The numbers of their offspring that grew to adults were scored. We censored 1–3 worms per experiment that died prematurely during the reproductive period (days 1–4 of adulthood) due to the vulva bursting, internal hatching of F1 offspring (bag of worms), or contamination of plates.

Gene sets

All gene sets are in Supplementary Data 1.

- Spermatogenesis genes (i.e., spermatogenesis-specific genes): RPKM in spermatogenic germlines (from either wild-type or him-8 males) > 15, RPKM in wild-type oogenic germlines < 1, and DESeq2 FDR < 0.05 for significance of higher expression in spermatogenic compared to oogenic germlines. Spermatogenic germlines from wild-type males compared to oogenic germlines identified 1369 genes, and him-8 males compared with oogenic germlines identified 1330 genes. The 1298 spermatogenesis-specific genes that overlapped between the two comparisons were used in our analyses. The 1298 spermatogenesis genes we defined included 690 of the 827 genes defined in Reinke et al.21. The 2498 spermatogenesis genes defined by Ortiz et al.26 included 1206 of our 1298 genes.

- Oogenesis genes (i.e., oogenesis-enriched genes) are as defined in Reinke et al.21 (Fig. 3) and Ortiz et al.26 (Supplementary Fig. 3).

- Sex-independent genes: RPKM in spermatogenic germlines (from either wild-type or him-8 males) > 5, RPKM in oogenic germlines > 5, FDR > 0.05, and log 2 (fold change) < 2 (genes with no significant differential expression between spermatogenic and oogenic germlines). Spermatogenic germlines from wild-type males compared with oogenic germlines identified 2549 genes, and spermatogenic germlines from him-8 males compared with oogenic germlines identified 2472 genes. The 2097 overlapping genes from these two comparisons were used in our analyses.

- Soma-specific genes (1171 genes) are genes expressed in at least 1 of 3 somatic tissues (muscle, gut, or neuron) with at least 8 SAGE tags57 but not enriched21 or detectably expressed58 in the adult germline.

- Silent genes are 410 serpentine receptor genes that are expressed in a few mature neurons and are not expected to be expressed in germlines, originally defined in23.

Statistics

Statistical analysis and sample sizes for all experiments are described in figure legends. Student’s t-test was performed using Excel. Mann–Whitney U-test and hypergeometric test were performed using R. At least 2 biological replicates of sequencing data (ChIP/MNase/RNA-seq) were obtained; sample sizes were based on experience and the standards in the field. We omitted sequencing data generated and analyzed during the optimization phase of the project. The final samples included were those generated after optimization. DESeq2 was used to test for differential expression by use of negative binomial generalized linear models. Adjusted p-values were calculated by DESeq2 using the Benjamini–Hochberg method.