Illumina HiSeq 2000 sequencing; ChIP-Seq for RNA Polymerase II in MCF7 breast cancer cells
ChIP-Seq for RNA Polymerase II in MCF7 breast cancer cells
MCF7 cells were cultured in RPMI1640 medium (GIBCO/BRL) supplemented with 10% (v/v) fetal bovine serum (GIBCO/BRL), 2.0 g/L sodium bicarbonate, and were incubated in a humidified 37 degrees C incubator with 5% CO2. Immunoprecipitation was performed according to the manufacturer's protocol (Upstate Biotechnology, Inc., Lake Placid, NY) with slight modifications. MCF7 cells were fixed for 10 min with 1% of formaldehyde at room temperature, and then quenched with a final of 1M of glycine. The cells were lysed and the chromatin was sonicated to 200-500 bp fragments with a Bioruptor (Diagenode, Sparta, NJ). Chromatin was immunoprecipitated by using RNA polymerase II (ab5408-100; Abcam) and Protein A Agarose beads. The beads were washed once with each washing buffer, including low salt immune complex wash buffer, high salt immune complex wash buffer, and LiCl immune complex wash buffer, and twice with 1X TE buffer. Precipitates were eluted with 1% of SDS and 100 mM of NaHCO3. The samples were heated at 65 degrees C for 6 hr in order to reverse cross-link, extracted with phenol/chloroform, ethanol-precipitated. The sequencing libraries were constructed from immunoprecipitated and input DNA using TruSeq ChIP Sample Preparation Kit (Illumina Inc., USA) according to the manufacturer's instruction. The fragmented DNA was end repaired following by addition 3'-A to the ends and ligation of adapters. The adapter-ligated DNA library was sized-selected (300-500 bp) on a 2% agarose gel and amplified by PCR for 16 cycles with the use of KAPA HiFi DNA Polymerase (Kapa Biosystems).
Experimental Factor: immunoprecipitate
PolII