Semi skirted pcr plates

1ug of Trizol-extracted RNA from gravid adult animals (see above) was used for Taqman assays. Small RNAs were reverse transcribed into cDNA using the Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems, 4366596). oma-1 and gfp small RNAs were then quantified by qRT-PCR using TaqMan Universal Master Mix II, no UNG (Applied Biosystems, 4440040) and custom TaqMan small RNA assays from Applied Biosystems (assay IDs: gfp: CSLJH0V, oma-1 probe 1: CSKAJ9W, oma-1 probe 2: CSLJIF4, oma-1 probe 3: CSMSGMC, oma-1 probe 4: CSN1ESK). qRT-PCRs were performed using the CFX Connect machine (Bio-Rad) and semi-skirted PCR plates (Bio-Rad, 2239441).

Total RNA was extracted from gravid adult animals using TRIzol Reagent (Life Technologies, 15596018). 2ug of total RNA was reverse-transcribed to generate first-strand cDNA using the Superscript III First-Strand Synthesis System (Invitrogen, 18080051) and random hexamers. Note: total RNA was heated with dNTPs and random hexamers to 65°C for 5 mins and immediately chilled on ice before proceeding with remaining cDNA synthesis steps. First-strand cDNA was then treated with RNAse H at 37°C for 20 mins. cDNA was then 1:100 (for oma-1 and histone RNA quantification) or 1:8 (for all other quantifications) and 2ul was used to quantify gene expression using iTaq Universal SYBR Green Supermix (Bio-Rad, 1725120) according to manufacturer’s instructions. qRT-PCRs were performed using the CFX Connect machine (Bio-Rad) and semi-skirted PCR plates (Bio-Rad, 2239441). All qRT-PCR data was normalized to quantification of nos-3, a germline-expressed gene. qRT-PCR primers are listed in Table S2.