GSM8326370: U2OS,  REXO4-D386A mutant stable expression cell line without Doxycycline added, input; Homo sapiens; ChIP-Seq
Briefly, for each IP, U2OS cells (four 100 mm dishes per biological replicate for sequencing) were washed twice with cold PBS and cross-linked with 1% formaldehyde for 10 min at room temperature and then quenched with 125 mM glycine for 5 min at room temperature. Cells were lysed sequentially with buffer1 (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TritonX-100) containing protease inhibitors including 1 mM PMSF (Amresco), 1 μg/ml Aprotinin (Targetmol) and 1× cocktail (Cwbio) and buffer2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl) containing protease inhibitors as described above on a rotative shaker at 4°C for 10 min. After centrifugation, cell pellets were resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl) containing protease inhibitors as described above, followed by sonicating to appropriate fragment (250~500 bp). After sonication, the lysate was centrifuged at 14000 rpm for 10 min to collect the supernatant. Next, 2% of the supernatant was saved as input and the remaining was diluted with NETN buffer containing protease inhibitors as described above. For ChIP-seq using tet-on stable expression cell lines of nuclease-dead mutant REXO4-D386A and RNaseH1-D210N, the samples were incubated with anti-Flag M2 affinity beads (Sigma-Aldrich) on a rotative shaker overnight at 4°C. Then the beads were washed with the following buffers successively: once with low salt buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA; 20 mM Tris-HCl, 150 mM NaCl), once with high salt buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl) and twice with TE buffer. The protein-chromatin complex bound to beads was eluted using elution buffer (10 mM Tris-HCl, 1% SDS, 1 mM EDTA) supplemented with protease K and incubated with input overnight at 65°C shaking to revert crosslinking. After sequential RNaseA and Proteinase K treatment, The DNA samples were purified using phenol chloroform extract assay. The precipitated DNA samples were subjected to library preparation. The library was prepared by using NEBNext Ultra II DNA Library Prep Kit (NEB).