NextSeq 550 sequencing; ChIP-seq for TP63 transcription factor in primary CLL cells
ChIP-seq for TP63 transcription factor in primary CLL cells
Primary CLL cells were cultured in RPMI-1640 medium with L-glutamine and sodium bicarbonide enriched with 10% Fetal Bovine Serum (FBS) Superior and 15μg/ml gentamicin at a density of 3x10^6 cells/ml and maintained at 37⁰C and 5% CO2 in a humidified atmosphere. Cells were fixed with 1% formaldehyde  for 10 minutes at RT, quenched with glycine and lysed using cell and nuclei lysis buffers. Chromatin was extracted and sonicated to a range of 200-1000 bp using a Covaris S220 sonicator.For the immunoprecipitation step, chromatin was precleared with 20 μl magnetic protein G beads per sample for 1h at 4°C in a rotating wheel and then incubated with monoclonal TP63 antibody. NGS libraries were prepared according to a previously published protocol (Ford et al., 2014, DOI: 10.1186/1756-0500-7-312), modified with the addition of the NEBNext Multiplex Oligos for Illumina.