ChIP-reChIP Analysis of Trf2 and Tbp

ChIP experiments were performed on sonicated or MNase digested chromatin as previously described. ChIP-reChIP was performed for Trf2 and Tbp according to the same protocol with minor modifications as follows. After first IP [anti Flag M2 resin (Sigma) or anti-TBP (ab28175, Sigma)] chromatin was eluted with a 3-fold smaller volume of elution buffer (1% SDS, 0.1 M NaHCO₃). For Trf2, the eluate was diluted 10 times with ChIP-dilution buffer (1.1%Triton X100, 1.2 mM EDTA, 16.7 mM Tris-Hcl-pH8.0, 167 mM NaCl) and re-ChIPed with the polyclonal anti-Trf2 antibody 1727. For Tbp the second ChIP was performed using or anti-TBP (ab51841, Abcam). ChIP-seq libraries were prepared and sequenced on an Illumina Hi-seq2500 as single-end 50-base reads. After sequencing, peak detection was performed using the MACS software54 (link) (http://liulab.dfci.harvard.edu/MACS/). Global clustering, meta-analyses and quantitative comparisons were performed using seqMINER and R (http://www.r-project.org/). The Taf7l ChIP-seq data is from GSE5080737 (link).