GSM1665997: SKmel147-xl-Input; Homo sapiens; ChIP-Seq
For BRD2 and E2F1, 1-3x10^7 cells (SK-mel147 and SK-mel147 stably expressing control or isoform-specific shRNAs) were cross-linked, with 1% formaldehyde for 10 min at 25°C. Cells were resuspended and lysed for 10min at 4°C with ChIP lysis buffer (50mM Hepes-KOH, pH7.6, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% Igepal, 0.25% TritonX), washed in ChIP wash buffer (10mM Tris-HCl, pH8, 200mM NaCl, 1mM EDTA, and 0.5mM EGTA) for 10min at 4°C and resuspended in ChIP shearing buffer (10mM Tris-HCl pH8, 100mM NaCl, 1mMEDTA, 0.5mM EGTA, 0.1% w/v deoxycholic acid, and 0.5% w/v Lauroylsarcosine). Cells were aliquoted in Covaris AFA microtubes (~6.6x10^6 cells in 130 ul per tube) and sonicated using a Covaris E220 (Intensity 2, DC 5%, CPB 200, duration 840sec). Fragment size (200-400bp) was evaluated using Agilent Bioanalyzer 2100. Samples were then diluted to 1.5ml, TritonX was added to a final concentration of 1%, and pre- cleared with 30ul of Dynal Magnetic beads for 20min at 4°C. The antibodies were coupled to the magnetic beads for 12 hours in blocking buffer (0.5% FBS in PBS), incubation was performed for 12- 16 hours. Beads were subsequently washed 5 times using RIPA buffer (50mM Hepes-KOH, pH7.6, 300mM LiCl, 1mM EDTA, 1% NP-40 (IGEPAL), 0.7% Na-Deoxycholate) and once with Tris-EDTA (TE) buffer (50mM NaCl). Precipitated chromatin was eluted in 200ul of Elution buffer (50mM Tris-HCl pH8, 10mM EDTA, and 1% SDS), after 2 hours incubation at 65°C. Reverse cross-linking was carried out for 12 hours at 65°C. Chromatin was then treated with RNase A, for 2 hours at 37°C, and Proteinase K, at 56°C for 4 hours. DNA was purified with phenol/chloroform extraction followed by Chloroform/H2O extraction and MiniElute PCR purification tubes. Total of 25ng ChIPed DNA was processed for library preparation from each sample. All enzymes used are from NEB unless stated otherwise. 1. DNA was End Repaired in 50ul reaction containing 40ul of DNA, 5ul T4 DNA ligase buffer with 10mM ATP, 2ul 10mM dNTP, 1ul Klenow DNA pol (1u/ul), 1ul T4 DNA pol and 1ul T4 PNK. The reaction was incubated for 30min at 20C. Products were purified in Qiagen PCR purification column in 35ul. 2. Adding over-hang A to the 3’ end in 50ul reaction containing 34ul DNA, 5ul 10X NEB buffer 2, 10ul 1mM dATP and 1ul Klenow exo-. The reaction was incubated for 30min at 37C. Products were purified in Qiagen PCR Mini elute purification column in 14ul. 3. Adapter ligation in 30ul reaction containing 13ul DNA, 15ul 2X T4 DNA ligase buffer, 1ul of 1:200 dilution of Illumina true seq DNA sample prep kit and 1ul quick T4 DNA ligase. The reaction was incubated for 15min at 25C. Products were purified in Qiagen Mini elute PCR purification column in 15ul. 4. Size selection. Ligated products were loaded onto 2% Agarose gel (1XTAE), and run for 1 hour at 120V. DNA was viewed on low-wave trans illuminator and size selected at the 300-400bp range using the Kb+ invitrogen DNA ladder. Products were purified in Qiagen PCR purification column in 25ul. 5. PCR amplification was done in 50ul reaction containing 25ul 2X master mix (2X Phusion HF, FINZYMES), 2ul PCR primers (illumina) and 23ul DNA. PCR program according to the illumina protocol. Products were purified in Qiagen Mini elute PCR purification column in 15ul. PCR products were then run on an Agilent DNA 1000 chip for quantification and submitted for sequencing on the Hi-Seq 2000 illumina platform.
GEO Accession
GSM1665997