title: GSM3031744: FlagPLAG1_ChIPSeq_1; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-seq, 1x10^8 cells K562 cells co-overexpressing Flag-PLAG1-S and USF2 were cross-linked in 1% PFA and excess PFA was washed with glycine, cells were lysed in RIPA buffer (50mM Tris-Cl pH7.45, 150mM NaCl, 0.1% SDS, 2% NP-40, 1% Sodium deoxycholate) containing protease inhibitors and nuclei were subjected to probe sonication. Two percent of each sample was used for Input sequencing. Chromatin was diluted in dilution buffer (50mM Tris-Cl pH7.45, 150mM NaCl) to get a final SDS concentration of 0.025% to assist in immunoprecipitation (IP). Twenty μg anti-USF2 (SantaCruz, #sc-293443) or anti-Flag (Sigma, F1804) and Protein G dynabeads were used for IP. The precipitated complex was washed in low (50mM Tris-Cl, pH 7.45, 250mM NaCl, 2mM EDTA, 0.1%SDS w/v, 1% Triton X-100 v/v) and high salt buffer (same as low salt buffer except use 500mM NaCl) and chromatin was eluted in TE buffer at 65°C before reverse cross-linking with Proteinase K (NEB, P8107S). Sequencing libraries were prepared using NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (NEB, E7645S) and sequencing as 50 bps single ended reads was performed on Illumina HiSeq 1500 at a depth of 55 million reads per input and 42.5 million reads per IP.