GSM8125710: ChIPseq, NELFCD, flav, aux; Homo sapiens; ChIP-Seq
HCT116 and C2C12 cells were crosslinked with 1% formaldehyde for 10 min, quenched with 125 mM glycine for 5 min, and washed with PBS. After collection by centrifugation, the fixed cells were frozen and stored at -80 C. The thawed cells resuspended with LB1 Buffer (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10 % Glycerol, 0.5 % NP-40, 0.25 % Triton-X, 1 mM PMSF, 10 mM DTT, 1x cOmplete EDTA-free (Roche)) and incubated on ice for 10 min. After collection by centrifugation, the pellet was resuspended with LB2 Buffer (20 mM Tris-HCl (pH7.5), 200 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1x cOmplete EDTA-free) and incubated on ice for 10 min. After centrifugation, the pellet was resuspended with LB3 Buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 % Triton-X, 0.1 % Na-Deoxycholate, 0.1 % SDS, 1x cOmplete EDTA-free) and incubated on ice for 10 min. Then, we repeated the centrifugation and resuspension with LB3 Buffer. For shearing chromatin, sonication was performed for 12 sec 6 times with amplitude setting at 17 % of the maximum amplitude using Sonifier 250D. Between each cycle, chromatin was collected by centrifugation. After the step of sonication, cell debris was removed by centrifugation at 19000 g for 15 min. 50 μl of Dynabeads Protein A or Dynabeads Protein G (Invitrogen) was washed with 5 mg/ml BSA/PBS twice and resuspended with BSA/PBS. 4 μg antibody was added to the beads, and the mixture was rotated for 3 h at 4 C. The beads was washed with BSA/PBS twice and with LB3 Buffer once and resuspended with LB3 Buffer. Chromatin from HCT116 and from C2C12 cells were mixed and incubated with antibody beads for 14 h at 4 C. The beads was washed with LB3 Buffer once, with LB3 High Buffer ( 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1 % Triton-X, 0.1 % Na-Deoxycholate, 0.1 % SDS) twice, with RIPA wash Buffer (50 mM HEPES-KOH (pH 7.4), 0.25 M LiCl, 1 mM EDTA, 0.5 % Na-Deoxycholate, 1 % NP-40) 3 times and with TE50 Buffer (50 mM Tris-HCl (pH 7.5), 10 mM EDTA) once. Chromatin bound to beads was eluted with Elution Buffer (50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1 % SDS). The eluates were incubated at 65 °C for 6 h to reverse crosslinks and then treated with RNaseA and then with proteinase K. The samples were purified with QIAquick PCR purification kit (QIAGEN).  Methods Mol Biol, 2017. 1515: p. 257-271). The library for sequencing was prepared with NEBNext DNA Library Prep Master Mix Set for Illumina and NEBNext Multiplex Oligos for Illumina (New England Biolabs).