GSM5032228: ctrl_4SU.mRNA-seq_rep1; Homo sapiens; RNA-Seq
RNA was extracted using the RNeasy kit (Qiagen, 74104) according to the manufacturer's instruction including the on-column DNase treatment. 4SU-residues biotinylation of 100 μg mammalian 4SU labelled RNA was 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 1 hour 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). 200ng total RNA was used for oligo(dT) purification. Libraries were prepared using the KAPA mRNA HyperPrep kit (Roche).
GEO
GSM5032228