title: GSM5510513: SMC3 pull down for PDX sample 634; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-seq was performed according to the SimpleChIP® Enzymatic Chromatin IP kit #9003 (Cell Signaling Technologies, Danvers, MA) procedure. The chromatin was sheared using the Biorupter Pico (Diagenode, Liege, Belgium; 7 cycles of 30' on, 30' off). After sonication, samples were divided and ~4x10e6 cells were used for each ChIP experiment. Antibodies were added to concentrations as recommended by the manufacturer and incubated overnight at 4°C under continuous rotation. Antibodies used are anti-CTCF (3418S) (Cell Signaling Technologies), anti-SMC3 (ab9263) (Abcam, Cambridge, UK) and normal rabbit IgG #2729 (Cell Signaling Technologies). DNA was pelleted with ChIP-grade Protein G Magnetic Beads #9006 (Cell Signaling Technologies) and washed according to the manufacturer's protocol. After reverse cross-linking, DNA was purified with spin columns from the SimpleChIP kit or the Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany). DNA concentrations were measured using the Qubit HS DNA sensitivity kit (ThermoFischer, Waltham, MA). Libraries were prepared using the NEXTflex™ Rapid DNA Sequencing Kit (Bio Scientific). Samples were PCR amplified, checked for size and the absence of adaptor dimers on 2% agarose gel. Barcoded libraries were sequenced for 75 bp at a single end using the Illumina NextSeq500 sequencer.