small-RNA-seq after GFP-dsRNA injection to WT anemones and Rpph treatment of RNA 24-hours old gastrula replicate2
Wild-type Nematostella zygotes were injected with dsRNA carrying GFP sequence then frozen in liquid nitrogen after 24 hours. RNA was extracted with Tri-reagent (Merck Millipore) according to manufacturer's protocol then treated with Rpph enzyme to remove 5'ppp. RNA was size selected for 18-30 nucleotides long sequences on urea-PAGE gel (Bio-Rad) and libraries were prepared with NEBNext Multiplex Small RNA Library Prep Set for Illumina kit (New England Biolabs) with modified protocol adapted to low RNA input (Plotnikova et al, 2019)