GSM8488161: EU_shNT_OHT_rep2; Homo sapiens; OTHER
RNA was then extracted and purified using the Quick-RNATM Miniprep Kit (Zymo Research, cat. #R1.fastq.gz055) according to the manufacturer's instructions. Nascent RNA was biotinylated and purified starting from 50 μg of total RNA. EU was coupled to a cleavable biotin-azide linker (Azide-PEG(3+3)-S-S-biotin) (kind gift from Thanos Halazonetis) using the Click-IT chemistry as shown below: 50 μg of RNA (40 μg of human EU-labelled RNA and 10 μg of murine EU-labelled RNA) were mixed with the same volume of 2X Click-IT reaction cocktail (171 mM Tris-HCl pH 8, 8 mM CuSO4, 200 mM sodium-L-ascorbate, 0,1 mM biotin–azide) and incubated for 30 minutes at RT with gentle shaking. RNA was precipitated and the EU-labelled RNA was then isolated using 50 μl of Dynabeads MyOne streptavidin C1 (Invitrogen, Cat. No. 65001) each sample, that were washed before use two times with Binding and Washing Buffer 1X (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.5% Tween-20), followed by two washes with solution A (0.1 M NaOH, 0.05 M NaCl) and two washes with solution B (0.1 M NaCl). After washing, the beads were resuspended to twice the original volume with Binding and Washing Buffer 2X. RNase OUT (1:100, Invitrogen, Cat. No. 10777-019) was added to RNA and then samples were heated at 70°C for 5 minutes and placed back on ice to remove secondary structures. Then EU-labelled RNA was mixed with an equal volume of washed beads (100 µL) and incubated for 30 minutes on a rotating wheel at room temperature. The RNA-beads complex was then washed three times with Binding and Washing Buffer 1X and once with RNase free-water. Finally, the EU-labelled RNA was eluted by incubating the streptavidin beads with 55 μl of 2% β -mercaptoethanol (Calbiochem, Cat. No. 444203) in 10 mM Tris-HCl pH. 8 for 1 h at room temperature. 5 μl of each sample were used for Qubit quantification (RNA HS assay kit; Thermofisher, Cat. No. Q32852). TruSeq Stranded Total RNA Gold (Illumina, Cat. No. 20020598) TruSeq Stranded Total RNA Gold (Illumina, Cat. No. 20020598)