GSM4396408: TAIL-seq exp #1 siTENT4 Set2; Homo sapiens; OTHER
Total RNAs were extracted from HepG2.2.15 cells and HFF cells by TRIzol reagent (Invitrogen, 15596018) and purified with RNeasy MinElute Cleanup Kit (Qiagen) according to manufacturer's instruction. TAIL-seq was conducted as previously described. In brief, ~50 μg of DNaseI (Takara, 2270A) treated total RNA (>200 nt) was used to deplete rRNA twice by Ribo-Zero kit (Epicentre, MRZH11124 [discontinued] for primary HFF library or Illumina, TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat, 20020596 for HepG2.2.15 library). The rRNA-depleted RNAs were ligated to the 3′ adaptor and partially fragmented by RNase T1 (Ambion). The fragmented RNAs were pulled-down with streptavidin beads (Invitrogen), 5′ phosphorylated and purified by 6% urea-PAGE gel (500–1,000 nt). The purified RNAs were ligated to the 5′ adaptor, reverse-transcribed and amplified by PCR. The libraries were sequenced by paired-end run (51×251 cycles) on Illumina platform (MiSeq) with PhiX control library v3 (Illumina) and spike-in mixture.