GSM1844664: SUDHL5_Treated_Input_2; Homo sapiens; ChIP-Seq
Fixed cells were incubated in lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.2 % NP-40) for 1 hour at 4 ˚C.  Chromatin was digested at a concentration of 25 x106 cells/mL with 250 U/mL MNase (Roche,10107921001) for 20 minutes at RT in MNase reaction buffer (55 mM Tris-HCl pH 7.5, 0.05 mM EDTA, 2.5 mM MgAc2, 12.5 % glycerol, 25 mM KCl, 4 mM MgCl2, 1 mM CaCl2).  The digestion was stopped with 10 mM EDTA.  The chromatin was then sonicated with a 550 Sonic Dismembrator (Fisher Scientific) on power 4, using cycles of 0.7 seconds on and 1.4 seconds off for a total processing time of 3 minutes.  Lysates were centrifuged at 21,000 x g for 10 min at 4 ˚C.  5 x106 cell equivalents were used for each ChIP assay, diluted to 1 mL with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 1.2 mM EDTA, 1.1% Triton-X 100, 167 mM NaCl, 0.01 % SDS).  Chromatin was pre-cleared for 2 hours at 4 ˚C with Protein A Dynabeads (Life Technologies,10001D) in blocking buffer (1x PBS, 0.5 % BSA, 0.1% Tween-20).  Beads were captured and pre-cleared chromatin was incubated with antibodies overnight at 4 ˚C.  Samples were then incubated with Protein A Dynabeads in blocking buffer for 2 hours at 4 ˚C.  Beads were washed 3x with RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate, 0.1 % SDS) and 1x with RIPA high salt buffer (10 mM Tris-HCl pH 8.0, 360 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate, 0.1 % SDS).  Bound chromatin was eluted with elution buffer (10 mM Tris-HCl, 1 % SDS, 300 mM NaCl, 10 mM EDTA pH 8.0).  Crosslinks were reversed by incubation at 65 ˚C for 6 hours.  Eluted chromatin was treated with 150 U/mL RNase A for 30 minutes at 37 ˚C and digested with 75 µg/mL proteinase K for 1 hour at 55 ˚C. ChIP DNA was purified with using a MinElute PCR Purification Kit (QIAGEN) and quantified with a Qubit fluorimeter (Life Technologies). NuGEN Ovation Ultralow DR multiplex system used to generate the libraries.  16 cycles of amplification was performed.  The libraries were size selected by agarose gel.  250-350bp fragments were taken.