GSM5032172: MRC5VA_HS60min_tc_TTchem-seq_rep2; Homo sapiens; RNA-Seq
RNA was extracted by TRIzol (Thermo Fisher Scientific, 15596026) according to the manufacturer's instructions. As a control for equal sample preparation, we spiked-in S. cerevisiae (strain BY4741, MATa, his3D1, leu2D0, met15D0, ura3D0) 4-thiouracil (4TU)-labelled RNA. S. cerevisiae were grown in YPD medium overnight, diluted to an OD600 of 0.1, and grown to mid-log phase (OD600 of 0.8) and labelled with 5 mM 4TU (Sigma-Aldrich, 440736) for 6 min. Total RNA was extracted using the PureLink RNA Mini kit (Thermo Fisher Scientific, 12183020) following the enzymatic protocol. For purification of 4SU labelled RNA, 100 μg mammalian 4SU labelled RNA was spiked-in 1/100 of 4TU-labelled S. cerevisiae RNA. The 101 μg RNA was fragmented by addition of 20 μL 1 M NaOH to fragment RNA and left on ice for 20 min. Fragmentation was stopped by addition of 80 μL 1 M Tris pH 6.8 and cleaned up twice with Micro Bio-Spin™ P-30 Gel Columns (BioRad, 7326223) according to the manufacturer's instructions. Biotinylation of 4SU-residues were carried out in a total volume of 250 μl, containing 10 mM Tris-HCl [pH 7.4], 1 mM EDTA and 5mg MTSEA biotin-XX linker (Biotium, BT90066) for 30 min at room temperature in the dark. RNA was then purified by phenol:chloroform extraction, denatured by 10 min incubation at 65°C and added to 200 μL μMACS Streptavidin MicroBeads (Miltenyi, 130-074-101). RNA was incubated with beads for 15 min at room temperature and beads applied to a μColumn in the magnetic field of a μMACS magnetic separator. Beads were washed twice with pull-out wash buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween20). 4SU-RNA was eluted twice by addition of 100 mM DTT and RNA cleaned up using the RNeasy MinElute kit (Qiagen, 74204). Libraries were prepared using the KAPA RNA HyperPrep kit (Roche) using 5 min 65°C fragmentation incubation to anneal primers but to prevent further fragmentation of the samples.
GEO
GSM5032172