GSM3873088
GSM3873088: 120_CTCF_ChIP_KO_Th1_CTCF_rep2; Mus musculus; ChIP-Seq
For ChIP-seq, 10 million Th1 and Th2 cells cultured as indicated above were cross-linked for 10 min with 1% formaldehyde and harvested. Cells were lysed by sonication (Sonifier S-450 Digital Ultrasonic Cell Disruptor/Homogenizer, Branson Ultrasonics) in shearing buffer (50mM Tris-HCl pH 7.6, 0.2% Triton X) and immuno-precipitated using a polyclonal rabbit anti-mouse anti-CTCF antiserum (07-729, Millipore Sigma, 8 µl per assay). ChIP was performed overnight at 4 ˚C using Protein A Dynabeads (Thermo Fisher). Antibody-bound beads were washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE buffer (pH 8.0) containing 0.2% Triton X-100, and once with TE buffer (pH 8.0). DNA was released by incubating the beads at 65°C for 4 hrs in the presence of 0.3% SDS and 1 mg/mL Proteinase K. ChIP DNA was purified by a DNA clean and concentrator column (Zymo research). 80 ng of ChIPed DNA was subsequently used to prepare ChIP-Seq libraries using Ovation SP Ultralow DR Multiplex system (Nugen) following the manufacturer's protocol. Libraries were sequenced for 50 single read cycles on HiSeq 3000 following the manufacturer's protocols.
GEO
GSM3873088
GEO Accession
GSM3873088