GSM8404798: WT1_TTseq; Mus musculus; RNA-Seq
The media was aspirated from cell plates and replaced with 10 mL of 500 nM 4-thiouridine (4sU, Carbosynth T4509) containing ES cell media and the plates incubated at 37°C with 5% CO2 for 5 min. After 5 min, the 4sU-containing media was aspirated and the cells were washed with 1xPBS, and 2 mL TRIzol (Invitrogen) was added directly to the plate. Cell lysate were collected, and RNA was extracted according to ThermoFisher's recommendations. RNA concentration was determined by Qubit with the Qubit RNA broad range quantification kit (ThermoFisher). 100 µg of total RNA was diluted to a concentration of 240 ng/µL at a volume of 416.67 µL in 1XTE and then fragmented with a Diagenode Bioruptor Pico on high power for one 30 second cycle. The fragmented RNA was then combined with 283.33 µL 1XTE, 100 µL 10X Biotinylation buffer (100 mM Tris pH 7.4 and 10 mM EDTA), and 200 µL of 1 mg/mL biotin-HPDP (ThermoFisher) in dimethylformamide (DMF; freshly prepared). Samples were vortexed, then incubated in a thermomixer at 37°C shaking at 1000 RPM in the dark for 2 hours. Samples were then chloroform extracted, isopropanol/salt precipitated, and resuspended in 22 µL of nuclease-free water. 60 uL of Streptavidin C1 beads (Invitrogen) were prepared for RNA separation as by washes with 1 mL of 1 M NaOH and 50 mM NaCl and resuspended in 60 µL of TT-seq Binding buffer (10 nM Tris pH 7.4, 300 mM NaCl, 0.1% Triton). Then, 60 µL of prepared streptavidin C1 beads were added to each sample and rotated at room temperature for 20 min. Following incubation, the samples were magnetized for 1 min and the supernatant (containing the unlabeled RNA) was placed in a separate 1.5 mL tube and put on ice. The unlabeled RNA from supernatant was Phenol:Chroloform:Isopropanol(PCI)/chloroform extracted, isopropanol/salt precipitated, and resuspended in 100 µL of nuclease-free water. The bead-bound labeled nascent RNA was washed twice with 500 µL of High Salt buffer (50 mM Tris pH 7.4, 2M NaCl, 0.5% Triton), twice with 500 µL of TT-seq Binding buffer, and once with 500 µL of Low Salt buffer (5 mM Tris pH 7.4, 0.1% Triton), rotating for 1 min at room temperature, re-magnetized and resuspending the beads during each wash. The nascent RNA was recovered from the beads through two rounds of 100 µL of freshly prepared 100 mM DTT and incubating in a thermomixer at 65°C and 1000 RPM shaking for 5 min. Eluted nascent RNA recovered with a PCI extraction and an isopropanol/salt/glycogen precipitation. RNA pellets were resuspended in 25 µL of nuclease-free water. Strand-specific nascent RNA library building was performed using the NEBNext Ultra II Directional Library kit according to the manufacturer's protocol with the following changes: The rRNA depleted RNA was fragmented at 94°C for fifteen minutes following the manufacturer's instructions for intact RNA, the fragmentation, first strand cDNA synthesis, and second strand cDNA synthesis were performed at double the reaction volume in the manufacturer's instructions, the adaptors were diluted 1:5 in Adaptor Dilution Buffer, the primers were diluted 1:5 in nuclease-free water, and 7 cycles of PCR were used to amplify the libraries. Finished libraries were quantified by Quibit with the dsDNA High Sensitivity kit and run on a Fragment Analyzer to confirm high quality of each library prior to sequencing. Libraries were sequenced paired-end on an Illumina NextSeq500 to ~40 million mapped reads.