GSM6257289: HEK293 shCtrl-20min TT-Seq; Homo sapiens; OTHER
RNAs were isolated using TRIzol reagent and fragmented in 166 mM NaOH on ice for 20 min.. To biotinylate 4SU-RNAs 3 μL of biotin buffer (833 mM Tris-HCl, pH 7.4, and 83.3 mM EDTA) and 50 μL of 0.1 mg/ml MTSEA biotin-XX linker (Biotium) were added to the 200 μL of fragmented RNA and incubated at RT for 30 min in the dark. Biotinylated RNAs were isolated by adding equal volume of phenol/chloroform/isoamyl alcohol (25:24:1 (vol/vol/vol)) and subsequently precipitated in NaCl/isopropanol. Biotinylated RNA fragments were then separated from non-biotinylated fragments using μMACS streptavidin MicroBeads (μMACS Streptavidin Kit, Miltenyi Biotec). library preparation using TruSeq ChIP Library Preparation Kit (Illumia). Libraries were prepared and indexed according to manufacturer's protocol. Indexed libraries were pooled and sequenced on an Illumina HiSeq 2500.