mNET-seq was performed using ~25 million K562 cells per sample. All buffers contained protease inhibitor cocktail. Briefly, the nuclei were extracted by incubating cells in HLB+N buffer (10mM Tris HCl pH7.5, 10mM NaCl, 2.5mM MgCl2, 0.5% NP40) for 10 minutes on ice, followed bycentrifugation(400g, 5min) through a sucrose cushion (HLB+N+ 10%sucrose). Nuclei were then resuspended in 125μl NUN1 buffer (20mM Tris HCl pH8.0, 75mM NaCl, 0.5mM EDTA, 50% glycerol) followed by addition of 1.2ml of NUN2 (20mMHEPESpH 7.6, 300mM NaCl, 0.2mM EDTA, 7.5mM MgCl2, 1% NP40, 1M urea), vortexing, and incubating for 15min on ice. The chromatin was pelleted by centrifuging the nuclear lysate at 16000g for 10min at 4°C, followed by resuspension in 100μl of (2μl NEB MNase + 98 μl MNase buffer (50mM TrisHCl pH 7.9, 5mM CaCl2, 250mM NaCl)). The chromatin was incubated for 2minutes at 37º in a thermoshaker (14000 RPM), and the reaction was stopped by addition of EGTA to 12.5mM and incubation on ice for 10min. Samples were centrifuged at 16000g for 10min and the supernatants were diluted to 1ml with IP buffer (50mM Tris HCl pH 7.5, 140mM NaCl,.05% NP40)+2ul murine RNAse inhibitor (NEB).Immunoprecipitationwas carried out with anti-pan pol II CTD (Invitrogen, MA1-26249) or Ser2P pol II CTD (Activ Motif, 61984) coupled to protein A/G magnetic beads for 1.5hours at 4ºC. IPs were then washed eight times with wash buffer low salt wash buffer (50mM Tris HCl pH 7.5, 140mM NaCl,.05% NP40). After washing, on-bead phosphorylation was performed in a 25μl reaction with 5 units T4 PNK 3'phosphatase minus (NEB), 1μl murine RNAse inhibitor in 50mM Tris HCl pH 7.5, 100mM NaCl, 5mM MgCl2, 1mM DTT, 0.01% NP40 at 37º for 5min. in a Thermomixer (14000 RPM). The beads were immersed in Trizol, and subsequently, 0.5 ng of DNAse-treated Drosophila RNA spike-ins were added to each sample. The RNA was extracted inTrizol(200μl) and libraries were generated with QIA miRNA seq kit