library_construction_protocol: Unless otherwise stated all RNA-seq experiments, beyond single embryo experiments (below) were conducted on adult animals. To generate rnst-2 mutant males, heterozygous rnst-2/tmc16 males and females were mated to generate progeny that's parents had equivalent genotypes. Individual ~50 F1 L4 from these crosses, propagated at 20°C, were then picked as males and females separately to NGM OP50 plates.  These animals were then developed at 20°C for 24 hours to generate adult animals. These animals were then picked into 1X M9 media with 0.5% polyethylene glycol (mPEG) in a 1.5 mL low adhesion microcentrifuge tube. Worms were washed three times with M9/mPEG and once with cold nuclease free water before resuspending in 1 volume 2X worm lysis buffer (1% SDS, 80mM Tris HCl, 400 mM NaCl, 20 mM EDTA, 0.8 mg/mL Proteinase K). The samples were then incubated at 55°C for 15 minutes while shaking and visually inspected to ensure complete lysis. Following worm lysis, 2X volume of TRI reagent (Invitrogen, AM9738) was added followed by 1/5th volume of BCP (catalog #). This solution was mixed thoroughly and transferred a Phase Lock Gel Heavy tube (QuantaBio, 2302830; pre-spun at 14,000 RPM for 1 minute) and spun at 14,000 RPM for 4 minutes at 4°C. The aqueous layer was then transferred to a new low-adhesion microcentrifuge tube, and 15 μg of GlycoBlue coprecipitant (Invitrogen AM9515) was added prior to the addition of 1.1 volume isopropanol. Samples were then precipitated at -20°C for at least 1 hours, and up to a month in isopropanol (longer storage times at -80°C) to purify total RNA. To generate small RNA and mRNA-seq libraries RNA samples in isopropanol were centrifuged at 14,000 RPM for 15 minutes at 4°C, then washed with cold 70% ethanol, spun again at 14,000 RPM for 5 minutes at 4°C, and finally reconstituted in nuclease-free water. RNA isolation from 50 adult male and females, yields ~500ng and ~1µg, respectively. For small RNA 200 ng of total RNA was used as input for all small RNA sequencing experiments with library construction carried out using a modified Illumina Tru-Seq small RNA library prep protocol (Illumina RS-200). Small RNAs (18 – 40 nt) were first purified by was size selection from denaturing 15% polyacrylamide 7M urea TBE gel electrophoresis. Small RNAs from eluted in 10mM Tris-HCl pH 7.5, 300mM NaCl, and 1mM EDTA shaking overnight at room temperature. Small RNAs were then precipitated by separating the gel debris using 0.45µM cellulose-acetate filters, adding 1ul GlycoBlue and 1.1 volume of isopropanol, mixing well, and placing the samples at -20°C for at least 1 hour. The samples then were centrifuged at 14,000 RPM for 15 minutes at 4°C and the supernatant was discarded before washing with 1X volume of cold 70% ethanol. Following a 14,000 RPM spin at 4°C for five minutes and after complete ethanol removal, the pellet was reconstituted in RNA 5´ Pyrophosphohydrolase (RppH - NEB M0356S) per manufacturers guidelines and incubated at 37°C for one hour. Following RppH treatment, small RNAs were precipitated and ligated to the 3´ adapter using Truncated T4 RNA ligase (Lucigen LR2D11310K). Small RNAs were then ligated to the 5´ adapter using T4 ligase, and subsequently reverse transcribed using Superscript II (Invitrogen 18064014) following the Tru-Seq small RNA protocol. Following PCR optimization, to determine the proper number of cycles for amplification, the libraries were amplified, gel purified on a 8% TBE polyacrylamide gel (to remove cloned products with no insert) and combined at equimolar concentration. Libraries were then sequenced using an Illumina NextSeq1000 (75-bp single read). For standard  mRNA-seq library construction was carried out using the Illumina stranded mRNA kit as per manufacturer's instructions (Illumina 20040534). In brief, poly-adenylated RNAs were captured using oligo(dT) magnetic beads from 200-800ng total RNA. The RNA was then fragmented, primed for cDNA synthesis and subsequently cleaned up using AMPure XP beads (Beckman Coulter, Cat# A63881). Following PCR optimization, to determine the proper number of cycles from amplification, the libraries were amplified and combined at equimolar concentrations before being purified using non-denaturing PAGE. Paired-end sequencing (50 bp per read) was performed using an Illumina NextSeq1000. For single embryo RNA-seq cross plates were prepared by placing 50 WT (fog-2(q71) L4-stage females and 50 rnst-2 Δ/Δ (pen1); fog-2(q71) L4-stage males on 60 mM NGM plates seeded with OP50. The next day mated females full of eggs were picked and washed several times with M9 mPEG and then subjected to a 5-minute hypochlorite treatment. Early-stage embryos, identified under the microscope, were then transferred to 10ug/ul chitanase in M9 mPEG treatment and incubated at room temperature for 8-10 minutes. Following chitinase treatment 2-cell and 8-cell embryos, identified visually under a dissecting microscope were placed in RNA worm lysis buffer in PCR tubes and incubated at 55°C for 10 minutes. Embryo lysates were then stored at -80°C before proceeding to library preparation. Single-embryo mRNA-seq libraries were constructed using the SMART-Seq protocol, used previously for single mouse preimplantation embryos. RNA was isolated with RNAClean-XP beads (Beckman Coulter, Cat# A63987) and full-length polyadenylated RNA was reverse transcribed using Superscript II (Invitrogen, Cat# 18064014). The cDNA was then amplified using 10 cycles and subsequently 0.33 ng of cDNA was used to construct a pool of uniquely indexed samples using the Nextera XT kit (Illumina, Cat# FC-131-1096). Finally, pooled libraries were sequenced on a NextSeq1000 with paired-end sequencing (50 bp per read).