GSM6857630: MCF10A_notreat_rep3; Homo sapiens; RNA-Seq
Total RNA was extracted using TRIzol. 100 μg of total RNA was added to a 250 μL MTS-biotin labeling reaction with 1X Biotinylation buffer (10mM HEPES, pH 7.5, 1 mM EDTA) and 5μg of MTSEA biotin-XX (Biotium) dissolved in 20% DMF. Reactions were nutated at room temperature for 30 min in the dark, then phenol/chloroform extracted, followed by ethanol precipitation. The RNA pellets were dissolved in 50 μL of dH2O and incubated at 65°C for 10 min followed by rapid cooling on ice. Biotinylated 4sU-labeled RNA was separated from unlabeled RNA using the μMacs Streptavidin Kit (Miltenyi). 100 μL of μMacs Streptavidin Microbeads were added to the 50 μL of RNA and nutated at room temperature for 15 min. Meanwhile, μMacs μColumns (Miltenyi) were equilibrated twice with 100uL of Equilibration Buffer for nucleic acid applications (Miltenyi). Samples were applied to the μColumns and flow-throughs were collected. The μColumns were then washed twice with 500 μL of high salt wash buffer (100mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween-20). 4sU-labeled RNA was eluted from the μColumns with 100 μL of freshly prepared 100 mM DTT. A second elution was performed 5 min later with another 100 μL of 100 mM DTT. The eluates were ethanol precipitated and RNA pellets were resuspended in 20 μL of dH2O. Sequencing libraries were constructed using the CORALL Total RNA-Seq Library Prep Kit with RiboCop (Lexogen).