GSM2214178: mTAIL-seq exp #8 wt-actegg(0-2hr); Drosophila melanogaster; RNA-Seq
Total RNAs were extracted from HeLa cells or Drosophila samples by TRIzol reagent (Invitrogen, 15596-018). Total RNA (~1–5 ug) was ligated to 3′ hairpin adaptor using T4 RNA ligase 2 (NEB, M0239) for overnight. 3′ ligated RNA was partially digested by RNase T1 (Ambion, AM2283) and subject to streptavidin beads (Invitrogen, 11206D). 5′ phosphorylation by PNK reaction (Takara, 2021B) and endonucleolytic cleavage by APE1 reaction (NEB, M0282) were performed on beads. Subsequently, RNA was eluted by 2X RNA loading dye and gel purified by 6% Urea-PAGE gel in the range of 300–750 nucleotides. The purified RNAs were ligated to 5′ adaptor, subjected to reverse-transcription (Invitrogen, 18080-085) and amplified by PCR using Phusion DNA polymerase (Thermo, F-530L). PCR products were purified by AMPure XP beads (Beckman, A63881).
GEO Accession
GSM2214178