GSM9737650: Liver, Ctrl, IgG; Mus musculus; OTHER
Nuclei were isolated from ~50 mg slow-frozen liver tissue by Dounce homogenization in nuclear isolation buffer (20mM HEPES-KOH pH 7.9, 20% glycerol, 10mM KCl, 0.5M spermidine, and 0.1% Triton X-100 with EDTA-free protease inhibitors). Nuclei were bound to activated concanavalin A–coated magnetic beads and incubated with primary antibody (anti-IRF3, Proteintech; anti-Pol II, anti-Pol II Ser5P, anti-Pol II Ser2P, all Cell Signaling Technology; IgG, EpiCypher) at 1 µg/mL overnight at 4°C with gentle rotation. Incubation with secondary antibody (1:50, anti-rabbit or anti-mouse; EpiCypher) was performed for 30 mins at room temperature, followed by incubation with protein AG-Tn5 transposase fusion (EpiCypher) at 1:20 for 1 hour at room temperature. Tagmentation was activated by adding MgCl₂ to a final concentration of 10 mM at 37°C for 1 hour. Reactions were stopped, and DNA was extracted by heating nuclei to 65°C in 0.1% SDS in 10mM TAPS buffer for 1 hour followed by column purification (Zymo Research). Libraries were amplified using 11 cycles of PCR with indexed primers (i5, 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTAT-3', and i7, 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGGAGATGTG-3') and size-selected by AMPure XP bead purification (Beckman Coulter) to enrich for fragments >150 bp. Libraries were quantified by Tapestation and Qubit and pooled at equimolar ratios.