GSM4909837: FTB2M_input_rep2; Homo sapiens; ChIP-Seq
Samples were homogenized using a Teflon homogenizer in H buffer [225 mM mannitol, 75 mM sucrose, 10 mM Hepes-NaOH (pH 7.8), 2 mM EDTA (pH 8); pH was adjusted to 7.6 with HCl; 2-mercaptoethanol was added prior to use]. Homogenates were centrifuged two times at 900 g for 10 min to remove nuclei and undisrupted cells. The supernatants were then centrifuged at 12,000 g for 10 min and pellets were resuspended in H buffer and centrifuged again. The obtained pellets were resuspended in H buffer, and formaldehyde was added to the suspension to a final concentration of 1%, and the solution was incubated for 15 min at r.t. The cross-linking reaction was quenched by adding glycine solution at a final concentration of 125 mM to the reaction mixture, followed by incubation for 5 min. Mitochondria were then collected by centrifugation at 12,000 g for 5 min. The pellets were resuspended in ice-cold H buffer and centrifuged at 12,000 g for 5 min.  mitochondria were lysed with IP buffer [150 mM KCl, 25 mM Hepes-KOH (pH 7.8), 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol, 0.5% Tween-20, and a proteinase inhibitor cocktail] and were sonicated using Bioruptor UCW-310. The resulting solution was centrifuged at 14,000 g for 5 min.  Supernatants were incubated with polyclonal rabbit antibodies against either TFB2M or POLRMT, and control normal rabbit IgG for overnight.  Subsequently, Protein G-magnetic beads were added to the mixtures and they were incubated for 1.5 h. After washing the beads with IP buffer three times, elution was performed by adding elution buffer [0.1M glycine-HCl (pH 2.5)] to the beads. Immunoprecipitation samples were reverse-crosslinked by incubating at 65 degree for 16 h, followed by proteinase K treatment. DNA was prepared by phenol-chloroform extraction and ethanol precipitation, and used for library construction using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). Sequncing was run on MiSeq in the 150-bp paired-end mode.