GSM4824146: S3 nascent RNA replicate 1; Mus musculus; RNA-Seq
Cells were  pelleted by centrifugation at 200G for 5 minutes and washed in PBS. The cell pellet was lysed in tri-reagent, and snap frozen on dry ice/ethanol. Total RNA was extracted from tri-reagent using a Direct-zol RNA kit (Zymo). 4sU labelled RNA was purified as previously described (Duffy et al. PMID 27925666). Briefly, 20-100µg of labelled total RNA was added to 50µl MTSEA-biotin-XX (0.1mg/ml in dimethylformamide) and 25μl of 10x Biotinylation buffer (100mM Tris pH 7.4 10mM EDTA) in a 250μl reaction and rotated for 30 minutes at room temperature. 400µl chloroform/isoamylalcohol (24:1) was added to the reaction and incubated for 3m. The aqueous phase was isolated using phase-lock gel tubes and purified by isopropanol precipitation. The RNA pellet was resuspended in 60µl RNase free water, denatured at 65°C for 10 min and cooled on ice for 5 min. RNA was incubated with 60µl µMacs streptavidin magnetic beads (Miltenyi) for 15 minutes at room temperature with rotation, applied to a MACS column in a magnetic stand and washed with 1ml MACS wash buffer (100mM Tris pH 7.5, 10mm EDTA, 1M NaCl, 0.1% Tween20) two times at 65°C and two times at room temperature. RNA was eluted from the column by applying 100 μL of freshly prepared 100 mM dithiothreitol (DTT), followed by a second elution round 5 min later, then purified using a Qiagen RNeasy RNA clean-up kit with on-column DNase digestion, eluting in 20μl of DEPC water. Libraries were prepared using the SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio) following the manufacturer's instructions with a fragmentation time of 3 minutes and 14 cycles of PCR amplification.