GSM4792395: melanoma cells input; Homo sapiens; ChIP-Seq
Cells were grown on 15cm plates and, once reached 80% of confluence, were fixed with PBS + 0.4% formaldehyde solution for 10min. Subsequently fixation reaction was stopped with 2M Glycin pH 8. Cells were then pellet and re-suspended in lysis buffer (EDTA 10mM, Tris HCl pH8 50mM, SDS 1%) and sonicate with Covaris E220 AFA power 200Hz 6 cycles 200s to get a DNA fragmentation between 500 and 200bp. Chromatin was then diluted in 60ug aliquots with 8 volumes of ChIP dilution buffer (Tris HCl pH8 16.7mM, EDTA 1.2mM, NaCl 167mM, Triton X-100 1.1%, SDS 0.01%). The immunoprecipitations were done incubating between 1-5ug of antibody overnight and the complex antibody-chromatin was then captured with G protein sepharose beads (Sigma Aldrich) 1h at 4°C. Beads were washed 2 times with Low Salt Buffer (Trish HCl pH8 20mM, EDTA 2mM, NaCl 150mM, Triton X-100 1%, SDS 0.1%), High Salt Buffer (Trish HCl pH8 20mM, EDTA 2mM, NaCl 500mM, Triton X-100 1%, SDS 0.1%), LiCl buffer (Tris HCl pH8 500mM, EDTA 1mM, Na deoxycholate 1%, NP40 1%, LiCl 0.25M), TE buffer (Tris HCl pH8 10mM, EDTA 1mM) and DNA was eluted 30min at room temperature with Elution buffer (NaHCO3 0.1M, SDS 1%). DNA was finally purified through phenol-chloroform and resuspend in 100uL of water. ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). ChIP-seq libraries were prepared from 1 to 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7 cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.