GSM9737660: Liver, IRF3-2D, fast, rep3; Mus musculus; OTHER
Nuclei were isolated from fresh liver tissue (~50–100 mg) by Dounce homogenization in ice-cold nuclear isolation buffer (10 mM Tris-HCl pH 7.4, 20% glycerol, 3 mM CaCl₂, 2 mM MgCl₂, 0.1% Triton X-100, 0.5 mM DTT, protease and RNase inhibitors). Nuclei were pelleted by centrifugation, washed, and resuspended in nuclear storage buffer (50 mM Tris-HCl pH 8.0, 25% glycerol, 5 mM MgCl₂, 0.1 mM EDTA, 5 mM DTT) at a concentration of ~10 × 106 nuclei/100 uL. Nuclear run-on was performed in the presence of biotin-11-NTPs (Perkin Elmer) at 37°C for 5 minutes. Reactions were stopped, and RNA was extracted using TRIzol. Nascent RNA was fragmented to ~100–200 bp by base hydrolysis (0.2 N NaOH, ice, 10 minutes) and enriched by streptavidin bead capture. Library construction was carried out following end-repair, ligation of barcoded adapters, and reverse transcription.