PHF3 regulates RNA stability through PHD and TLD domains - ChIPseq CNOT
PHF3 regulates RNA stability through PHD and TLD domains - ChIPseq CNOT
CNOT1 ChIP was performed in two biological replicates. 108 cells in 50 mL PBS were fixed for 10 minutes using 1% formaldehyde. After quenching by addition of 0.6M Glycine pH 3 for 15 minutes, cells were centrifuged and washed twice in cold PBS. Nuclei from108 fixed cells were isolated by rotating 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 protease inhibitor) for 10 min at 4°C. Nuclei were washed in 5 mL cold lysis buffer 2 (10 mM Tris-Cl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x protease inhibitors) for 10 min at room temperature. The pellet 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 protease inhibitors). Chromatin was sheared to an average size of 200-600 bp using the Bioruptor Pico (Diagenode) for 20 cycles, 30 sec on/30 sec off. Triton X-100 was added to a final concentration of 1% and 2.5% of mouse chromatin was added as a spike-in control. 1 mL chromatin (from 33 million cells) was mixed with 10 µg anti-CNOT1 antibody (Proteintech) and rotated ON at 4°C. Protein A Dynabeads (Invitrogen) were washed three times in cold block solution (0.5% BSA in PBS), antibody-bound chromatin was added to the beads and rotated 4-6 hours at 4°C. Beads were washed 5 times in RIPA washing buffer (50 mM Hepes/KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) and once in 50 mM NaCl in TE. Crosslinked 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. Crosslinks in eluted protein-DNA complexes were reversed at 65°C ON. RNA was degraded by adding 0.2 mg/mL RNase A for 2 h at 37°C, proteins were digested by adding 0.2 mg/mL proteinase K and 5.25 mM CaCl2 for 30 min at 55°C. DNA was purified by phenol-chloroform extraction, ethanol-precipitated and resuspended in 50 µL nuclease-free water. Next generation sequencing libraries were prepared using the NEBNext Ultra II DNA library Prep Kit for Illumina and NEBNext Multiplex Oligos Primer Set 1 (New England Biolabs) according to the manufacturer's instructions.