GSM8887742: ChronicMEKi, Rep2; Mus musculus; RNA-Seq
Nascent RNA was labeled in live cells and isolated using the Click-iT™ Nascent RNA Capture Kit (Thermo Fisher, Cat. No. C10365) following the manufacturer's protocol. Briefly, 1 × 10⁶ Tcells were seeded in 500 µL of T-cell media per well in a 12-well plate. Actinomycin D (100 pM) was added to the negative control wells for 12 hours prior to labeling. Cells were then incubated with 0.2 mM 5-ethynyl uridine (5-EU) (Lumiprobe, #2439) at 37°C for 30 minutes to label newly synthesized RNA. After harvesting, cells were centrifuged at 250g for 5 minutes, and total RNA was isolated using the Direct-zol RNA Miniprep Kit (Zymo Research, #R2050). For sequencing nascent RNA, the 5-EU-labeled nascent RNA was biotinylated through a click reaction with biotin-azide for 1 hour at room temperature, bound to Dynabeads MyOne Streptavidin T1 magnetic beads, and isolated for cDNA synthesis. Reverse transcription was performed using the SuperScript VILO kit (Thermo Fisher, Cat. No. 11754050). The resulting cDNA was purified and sequenced. For quantification in a limited number of cells, 5-EU-labeled nascent RNA was clicked to Alexa 488-azide and measured by flow cytometry. RNA was isolated from T cells using the Direct-zol RNA Miniprep Kit (Zymo Research, #R2050) following the manufacturer's instructions. RNA quantity and integrity were assessed using a NanoDrop spectrophotometer and Agilent Bioanalyzer, respectively, before sequencing.  Core Nascent RNA-seq samples were sequenced using the Illumina NextSeq 500 platform, generating 100-base pair paired-end reads.  Library preparation and sequencing were performed by the MSK Integrated Genomics Operation.