GSM4743285: Med14_msAuxin_Nutlin_Input; Homo sapiens; ChIP-Seq
Cells were fixed with 1% FA in PBS for 15 min. Crosslinked cells were resuspended in lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitors) and rotated for 10 minutes at 4°C. The pellet was sonicated on a Misonix 3000 sonicator for 10 cycles at 30 s each on ice (18-21 W) with 60 s on ice between cycles. Input material was reserved and the remainder was incubated overnight at 4°C with magnetic beads bound with MED1 antibody (Bethyl #A300-793A). DNA was eluted off the beads by incubation with agitation at 65°C for 15 minutes in elution buffer. Cross-links were reversed for 12 hours at 65°C. Eluted DNA was treated with RNase A (37°C for 2 hours) and proteinase K (55°C for 30 min). DNA was purified using Qiagen PCR purification kit. Purified immunoprecipitated DNA were prepared for sequencing according to a modified version of the Solexa Genomic DNA protocol. Fragmented DNA was end repaired and subjected to 18 cycles of LM-PCR using oligos provided by Illumina. Amplified fragments between 150 and 300bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence) were isolated by agarose gel electrophoresis and purified.