RNA extractions were performed using Ambion RNAqueous Total RNA kit (AM1931), including an on-column DNase I treatment using Qiagen DNase I (#79254). Total RNA was analyzed using a Bioanalyzer (Agilent) to check for RNA integrity, with the eukaryotic total RNA-pico program. RNA input for library construction was ∼30 ng. Small RNA libraries were made using the NEXTflex small RNA-seq kit v3 (PerkinElmer NOVA-5132-05), with the following modifications. One-quarter dilution of adapters was used. The 3′ adapter ligation step was done overnight at 20°C. Zygote libraries were amplified at 24 cycles. Postfertilization ovary libraries were amplified at 20 cycles, as prefertilization ovaries (Li et al. 2020 (link)). The library product was size-selected using PippinHT (Sage Science) 3% agarose gel cassettes.
Protocol detail
Small RNA Sequencing Library Preparation
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Corresponding Organization :
Other organizations : University of California, Davis, University of Georgia, University of Oklahoma
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Variable analysis
independent variables
- RNA extraction method (Ambion RNAqueous Total RNA kit)
- RNA integrity (analyzed using Bioanalyzer)
- Small RNA library construction (NEXTflex small RNA-seq kit v3)
- DNase I treatment (using Qiagen DNase I)
- RNA input for library construction (~30 ng)
- Adapter dilution (one-quarter dilution)
- 3' adapter ligation (overnight at 20°C)
- Amplification cycles (24 cycles for zygote libraries, 20 cycles for postfertilization ovary libraries)
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