GSM5018587: U2OS cells - HighMYC_MYC rep1_ChIP; Homo sapiens; ChIP-Seq
Cells were fixed in 1% formaldehyde and sonicated using the truChIP Chromatin Shearing Kit (Covaris) on the ME220 Focused-Ultrasonicator (Covaris) according to the manufacturer's protocol. 3.5µg of each antibody was rotated with 15µl of Dynabeads Protein G magnetic beads (Invitrogen) at 4℃ overnight and washed thrice with Beads Wash Buffer (0.1% Triton X-100 in PBS) to remove unbound antibodies. Antibody-bound beads were combined with sonicated chromatin from 5 million cells and rotated at 4℃ overnight. Beads were then washed thrice with Shearing Buffer D3 (Covaris), once with High Salt Washing Buffer (50mM HEPES pH 7.5, 350mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS), once with Lithium Chloride Wash Buffer (10mM Tris pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% Sodium Deoxycholate) and once with Tris-EDTA buffer. Chromatin was eluted from the magnetic beads in 100µl elution buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS) supplemented with 2µl of 0.5mg/ml RNase A (QIAGEN) at 55℃ for 2 hours. Chromatin was then decrosslinked with the addition of 2µl of 20mg/ml proteinase K (Ambion) at 55℃ for 4 hours and 37℃ overnight. For total input, chromatin from 500,000 cells were treated with RNase A and decrosslinked similarly. DNA was purified using the MinElute PCR Purification Kit (QIAGEN). ChIP-seq libraries were prepared using the ThruPLEX DNA-seq Kit (Rubicon) and sequenced paired-end 2 x 100bp on the HiSeq 2500 (Illumina).