GSM1631487: Input Control 30 mins Estradiol; Homo sapiens; ChIP-Seq
Cells were fixed with formaldehyde (1% (v/v), Sigma-Aldrich Inc.), neutralised with glycine, washed with 1XPBS and trypsinised (0.05% trypsin in 0.53mM EDTA (Invitrogen Inc. 25300-054). Cells were collected and lysed with cell lysis buffer +PI, collected and resuspended in nuclear lysis buffer +PI. Chromatin was sonicated to between 500-1000bp, 14x15sec cycles, and 5% was removed as “input”. 10.6μg of polyclonal anti-TOP2B antisera was added to protein A magnetic beads (Invitrogen, 100.02D). Chromatin was diluted, incubated with the beads and rotated at 4°C for 4.5hrs. IP’s were washed, Low Salt Buffer, High Salt Buffer, LiCl Buffer and TE Buffer respectively, resuspended in ChIP Elution Buffer+Proteinase K, rotated at 65°C for 2 hours followed by incubation at 95°C for 10 minutes. DNA was then purified using spin columns. 6-21ng/μl of DNA was sent for Illumina ChIP Sequencing.
GEO Accession
GSM1631487