title: GSM7213946: Treg, CTCF ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: 10 million Treg or Tcon cells were fixed in 1% formaldehyde for 10 min at room temperature, followed by the addition of glycine to quench the excess crosslinker. Cells were then washed in cold PBS, pelleted, flash-frozen in liquid nitrogen and  stored at −80 °C. Cell pellets were thawed on ice and incubated in cell lysis buffer (50 mM Hepes-KOH, pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100) for 10 min. The isolated nuclei were washed with wash buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.5 mM (ethylenebis(oxonitrilo)) tetra-acetate, 200 mM NaCl) and resuspended in shearing buffer (0.1% sodium dodecylsulfate (SDS), 1 mM EDTA, 10 mM Tris-HCl, pH 8). Nuclear chromatin was sheared in a Covaris E229 sonicator for 10 min to generate DNA fragments between ~200 bp and 1,000 bp. After clarification of insoluble material by centrifugation, the chromatin was incubated with CTCF antibody overnight at 4 °C. The next day, the antibody-bound DNA was incubated with Protein A + G Dynabeads (Invitrogen, catalog no. 10015D) in ChIP buffer (50 mM Hepes-KOH, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), washed and treated with proteinase K and RNase A. Crosslinking was reversed by incubation at 55 °C for 2 h. Purified ChIP DNA was used for library generation (NuGen Ovation Ultralow Library System v.2) according to the manufacturer's instructions. ChIP–seq libraries were sequenced by an Illumina HiSeq2500 sequencer.