title: GSM4198492: NaB_3h_R1_rep2; Homo sapiens; OTHER
library_construction_protocol: Following bromouridine labeling, cell were immediately collected by centrifugation and lysed in 1 mL TRIzol (Invitrogen).  RNA was extracted per TRIzol protocol. Goat anti-mouse IgG magnetic Dynabeads (110.33; Invitrogen) were prepared by washing 50 µL of beads with 200 μl 0.1% BSA in DEPC-PBS three times. Beads were resuspended in 200 μl 0.1% BSA in DEPC-PBS, followed by incubation with 2 μg anti-BrdU mouse monoclonal antibody (555627; BD Biosciences) and 20 U RNaseOUT (10777-019; Invitrogen) for 1 hour at room temperature. Beads were washed three times and resuspended in 200 μl 0.1% BSA in DEPC-PBS containing 20 U RNaseOUT. To isolate BrU labeled RNA, samples were heated at 80°C for 10 min, placed on ice, and 90% of the sample was added to prepared beads and incubated with rotation for 1 hour at room temperature. Nascent RNA bound beads were washed three times with 0.1% BSA in DEPC-PBS. For elution, beads were resuspended in 40 μl DEPC-water and incubated at 95 °C for 10 min, followed by magnetic capture. Supernatant containing purified nascent BrU-containing RNA was removed and quantified (Nanodrop; Thermo Scientific). cDNA libraries were prepared using reagents in the Illumina TruSeq RNA Prep Kit. Bru-labeled RNA was mixed with first strand buffer and random primers and fragmented by heating at 85 ̊C for 10 minutes. The first strand cDNA was then synthesized, in the presence of Actinomycin D, to result in strand specific reads. After purifying the first strand cDNA using AMPure RNAclean beads (Beckman Coulter), the second strand cDNA was synthesized. The resulting cDNA was purified with AMPure XP beads, followed by cDNA end repair, adenylation, and adapter ligation (TruSeq RNA Prep Kit). The samples were then run on a 3% agarose gel and size-selected by excising gel slices in the 300bp region. These gel slices were purified using the QIAEX II Gel Extraction Kit (Qiagen) and then DNA fragments were enriched using the PCR reagents from the TruSeq RNA Prep Kit. After a final purification using AMPure XP beads, the quality and concentration of the DNA libraries were determined using an Agilent Bioanalyzer. Bru-seq