title: GSM3873068: 119_Input_WT_Th1_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: 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.
attributes.tag: GEO Accession
attributes.value: GSM3873068