ChIPs were performed as described previously (5).
Mouse livers were minced finely in cold phosphate-buffered saline (PBS) and cross-linked in 1% formaldehyde for 10 min while rotating. Cross-linking was quenched by adding glycine to a final concentration of 0.125 M for 5 min while rotating. The tissue was rinsed in cold PBS and homogenized with a Dounce homogenizer in cold cell lysis buffer (10 mM Tris–Cl, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) and protease inhibitors. Cells were incubated at 4°C for 5 min to release nuclei. Nuclei were centrifuged at 13 000 g for 5 min to form a pellet. The pellet
was resuspended in nuclear lysis buffer [1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 50 mM Tris–Cl, pH 8.1) and protease inhibitors and sonicated using the Diagenode Bioruptor for 10 min on high, using 30 s intervals. Debris were removed by centrifugation at 13 000 g for 10 min, and the supernatant was collected and snap frozen in liquid nitrogen. A 10 μl aliquot was reversed by the addition of NaCl to a final concentration of 192 mM, overnight incubation at 65°C, and purification using a PCR purification kit (Qiagen, CA, USA). The chromatin concentration was
determined using a NanoDrop 3.1.0 nucleic acid assay (Agilent Technologies, Santa Clara, CA, USA)
Mouse livers were minced finely in cold phosphate-buffered saline (PBS) and cross-linked in 1% formaldehyde for 10 min while rotating. Cross-linking was quenched by adding glycine to a final concentration of 0.125 M for 5 min while rotating. The tissue was rinsed in cold PBS and homogenized with a Dounce homogenizer in cold cell lysis buffer (10 mM Tris–Cl, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) and protease inhibitors. Cells were incubated at 4°C for 5 min to release nuclei. Nuclei were centrifuged at 13 000 g for 5 min to form a pellet. The pellet
was resuspended in nuclear lysis buffer [1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 50 mM Tris–Cl, pH 8.1) and protease inhibitors and sonicated using the Diagenode Bioruptor for 10 min on high, using 30 s intervals. Debris were removed by centrifugation at 13 000 g for 10 min, and the supernatant was collected and snap frozen in liquid nitrogen. A 10 μl aliquot was reversed by the addition of NaCl to a final concentration of 192 mM, overnight incubation at 65°C, and purification using a PCR purification kit (Qiagen, CA, USA). The chromatin concentration was
determined using a NanoDrop 3.1.0 nucleic acid assay (Agilent Technologies, Santa Clara, CA, USA)
Cold spring harbour
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