NextSeq 550 sequencing; The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators - ChIP-seq
The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators - ChIP-seq
Cells were resuspended in 50 mL PBS/108 cells and fixed by adding 1% formaldehyde for 10 min. Formaldehyde was quenched by addition of 0.6 M glycine pH 3 for 15 min. Cells were washed twice in cold PBS.  Nuclei were isolated by resuspending the cells in 5 mL cold lysis buffer 1 (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 0.25% Triton X-100, 1x Complete protease inhibitors (Roche)) per 108 cells and rotating at 4°C for 10 min. After centrifugation nuclei were resuspended in 5mL/108 cells cold lysis buffer 2 (10 mM Tris-Cl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Complete protease inhibitors (Roche)) and rotated for 10 min at room temperature. The pellet after centrifugation was resuspended in 3 mL lysis buffer 3 (10 mM Tris-Cl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1x Complete protease inhibitors (Roche)) and chromatin was sheared to an average size of 200-600 bp by sonication for 20 cycles 30 sec on/30 sec off using Bioruptor Pico (Diagenode). 1% Triton X-100 was added after sonication. 5-10% of chromatin was kept as an input control for qPCR. For ChIP-seq 1.5% spike-in chromatin from a mouse cell line expressing endogenously tagged PHF3-GFP was added. Anti-GFP antiserum (Abcam ab290) was added to sheared chromatin and rotated ON at 4°C. Protein A Dynabeads (Invitrogen) were washed in cold block solution (0.5% BSA in PBS) three times, mixed with antibody-bound chromatin and rotated for 4-6 h at 4°C. Beads were washed in RIPA washing buffer (50 mM Hepes/KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) five times and in 50 mM NaCl in TE once. Immunoprecipitated protein-DNA complexes were eluted in 200 µL elution buffer (50 mM Tris-Cl pH 8, 10 mM EDTA, 1% SDS) for 15 min at 65°C. Eluates were incubated ON at 65°C to reverse crosslinks, treated with 0.2 mg/mL RNase A for 2 h at 37°C and 0.2 mg/mL proteinase K and 5.25 mM CaCl2 for 30 min at 55°C. DNA was purified by phenol-chloroform extraction followed by ethanol precipitation and resuspended in 50 µL nuclease free water.   ChIP-seq libraries were prepared using NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs) and NEBNext Multiplex Oligos for Illumina (New England Biolabs) according to the manufacturer's instructions.