PANDORA small-RNA-seq after GFP-dsRNA injection to actin::GFP anemones 24-hours old gastrula replicate1
Nematostella actin::GFP 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. Total RNAs were separated by 10% Urea-PAGE gel and 15-50nt small RNAs were recovered from the gel. Recovered 15-50nt sncRNAs were treated by T4PNK enzyme (New England Biolabs) to repair the 3'termini first; sncRNA was extracted with TRIzol reagent (Invitrogen) from the T4PNK reaction mixture. Purifed T4PNK-treated sncRNA was then treated by AlkB enzyme to remove RNA modification that block the reverse-transcription; sncRNA was extracted with TRIzol reagent (Invitrogen) from AlkB reaction mixture. The cDNA libraries of T4PNK-AlkB-treated sncRNA were prepared with NEBNext Small RNA Library Prep Set for Illumina kit (New England Biolabs).