title: GSM3045646: 4sURNA_60_R1; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted using phenol:chloroform:isoamyl alcohol (Sigma, P3803-400ML). Equal volume of biotinylated RNA and prewashed DynabeadsTM MyOneTM Streptavidin T1 beads (Thermo Fischer, 65601) were added to 2X B&W Buffer (10 mM Tris-HCl pH7.5, 1mM EDTA, 2M NaCl (Thermo Fisher, #65601)) and incubated at room temperature for 15 minutes under rotation. The beads were then separated from the mixture using DynaMagTM-2 Magnet (Thermo Fisher, #12321D) and after removal of supernatant, washed with 1X B&W three times. Biotinylated RNA was recovered from the supernatant after 1 minute incubation with RTL buffer (RNeasy kit, Qiagen, #74104) and purified using the RNeasy kit according to manufacturer's instructions. Total RNA libraries were prepared from 10 ng of DNAse treated total RNA and newly transcribed RNA using Ovation® RNA-Seq and sequenced on  Illumina HiSeq 2500.