G2 and metaphase arrested HeLa S3 cells were collected and fixed in 1xPBS containing 1% formaldehyde for 10 minutes. Glycine was added to a final concentration of 0.125M to stop cross-linking and cells washed in cold 1xPBS. Intact nuclei from ~ 5x106 cells were extracted with 1ml of Farnham lysis buffer and finally resuspended into 300ml of RIPA buffer. Nuclei were fragmented using a Qsonica sonicator for 5 minutes (30 seconds on/off, 40%). The efficiency of fragmentation was measured as the enrichment of ~ 300 bp fragments, tested on an agarose gel, and the DNA quantified in a Biodrop. To perform immunoprecipitations Dynabeads M-280 Sheep Anti-Rabbit (Invitrogen 11203D) were coupled to 5 mg of pS2 or IgG rabbit antibodies and incubated with 25 mg of fragmented chromatin samples overnight at 4°. Beads were then washed with LiCl wash buffer and eluted in 200 ml of IP elution buffer. Cross-link was reversed by incubating beads at 65° for 1 hour, supernatant (DNA) recovered after centrifugation and incubated at 65° for 14h. In parallel, input samples (25 mg of fragmented chromatin) were incubated at 65° for 14h and then purified using QIAquick PCR Purification Kit (QIAGEN).  For ChIP-seq, DNA recovered from pS2 IP and ~ 10 ng of sonicated INPUTs were used for library preparation NEBNext ChIP-Seq kit (NEB6240S)
GEO Accession
GSM4147297