Illumina HiSeq 2500 paired-end Sequencing of KAS20000485 (KAE20000470)
missing
We crosslinked1-2 x 106human bladder tumor cells for 5 min at 37 degree Celsius with formaldehyde at a final 1-2 We then carried out ChIP using previously described methods.80 In brief, we pre-cleared sonicated chromatin in RIPA buffer 20 mM Tris-Cl pH 8.0, 2 mM EDTA, 1-2 mM NaCl, 1-2Triton X-1-2, 0.01-2SDS with 2 microgram of rabbit IgG Cell Signaling Technology and 20 microliter of Protein G magnetic beads Thermo Fisher Scientific. We then immunoprecipitated pre-cleared chromatin using 2 microgram of FOXA1-2ntibody Abcam and 20 microliter of Protein G magnetic beads Thermo Fisher Scientific overnight at 4 degree Celsius with rotation. The next day, we washed the pulled chromatin, and we reverse crosslinked it with proteinase K 20 mg/ml and 1-2 SDS overnight at 65 degree Celsius. We extracted ChIPed DNA with phenol/chloroform and eluted it in 20 microliter of TE. We constructed ChIP-seq libraries using the ACCEL-NGS 2S PLUS DNA LIBRARY KIT Swift Bioscience, following the manufacturer's instructions. In brief, purified ChIP DNA was subjected to end-repairing and adaptor ligation, and applied 1-2cycles of PCR amplification. Amplified DNA was size selected and quantified using Bioanalyzer Agilent.
KOBIC
KAE20000470
Pohang University of Science and Technology
DRX602011