Chromatin Immunoprecipitation of HA-Tagged Proteins

Samples were cross-linked in 3% formaldehyde solution in PBS, and cross-linking was quenched with 0.2 M glycine. Nucleus enrichment was performed as described (Fiil et al. 2008 (link)). Samples were sonicated in lysis buffer (50 mM Tris-HCL at pH 8, 10 mM EDTA, 1% SDS) and further processed as described (Stracke et al. 2010 (link); Binkert et al. 2014 (link)). The chromatin was immunoprecipitated with anti-HA antibody (ChIP-grade; Abcam, ab9110) overnight at 4°C, after which cross-linking was reversed for 2 h at 85°C. DNA was purified using QIAquick PCR purification kit (Qiagen) before analysis with a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific) and the following primer sets: Pro_{FT_-100}-Fw (5′-AGAGGGTTCATGCCTATGATA C-3′), Pro_{FT_-100}-Rv (5′-CTTTGATCTTGAACAAACAGGTG-3′) (Bu et al. 2014 (link)), Pro_{FT_-1185}-Fw (5′-TTATCCTGGTCGTGCAAATG-3′), and Pro_{FT_-1185}-Rv (5′-CAAGCGGCCATATTATGGAA-3′) (Song et al. 2012 (link)). qPCR data were analyzed according to the percentage of input method (Haring et al. 2007 (link)). Each reaction was performed in technical triplicates; data shown are representative of three independent biological repetitions.