Multi-RNA Sequencing from 4-cell Embryos

RNA was isolated using Trizol or LS Trizol (Invitrogen). Total RNA samples were fractionated into small RNA (<200 nt) and long RNA (>200 nt) using the Monarch RNA cleaner (New England Biolabs). Small RNA libraries were prepared using the Small RNA-Seq Library Prep Kit (Lexogen). Both 5’-phosphate and 5’-phosphate independent libraries (RppH-treated)^{124 (link)} were prepared. Libraries from 4-cell embryos were also prepared using 18–30 nt gel-purified RNA using the SMARTer smRNA-Seq Kit (Clontech) and NEXTflex-Small-RNA-Seq (New England Biolabs) with similar results. RNA > 200 nt was treated with TURBO DNase (Ambion) and rRNA depletion carried out using RiboCop rRNA Depletion Kit for Human/Mouse/Rat (Lexogen). Long RNA (>200 nt) libraries were made using CORALL Total RNA-Seq Library Prep Kit (Lexogen). Both small and long RNA libraries were sequenced 150 nt from both ends using the Illumina NovaSeq 6000 System.