title: GSM2066633: LAN6-Input_control-Native_ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Native ChIP: Native ChIP was performed essentially as described (Hasson et al., Nat Struct Mol. Biol, 2013) with minor changes. Briefly: 20 million cells were gently trypsinized and resuspended in 2 ml of ice-cold buffer I (0.32 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mM PMSF and 1:1,000 protease inhibitor cocktail (Sigma)). Ice-cold buffer I supplemented with 0.1% IGEPAL (2 ml) was added, and samples were placed on ice for 8 min. The resulting 4 ml of nuclei was gently layered on top of 8 ml of ice-cold buffer III (1.2 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mM PMSF and 1:1,000 protease inhibitor cocktail) and centrifuged at 10,000g for 20 min at 4°C. Pelleted nuclei were resuspended in Native buffer A (0.34 M sucrose, 15 mM HEPES, pH 7.4, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 1 mM DTT and 1:1,000 protease inhibitor cocktail (Sigma)) to 400 ng/μl. MNase (Affymetrix) digestion reactions were carried out on chromatin (~100 μg), using ~0.1 U/μg chromatin in Native buffer A supplemented with 3 mM CaCl2 for 10 min at 37 °C. The reaction was quenched with 5 mM EGTA. The chromatin was resuspended in 10 mM EDTA, pH 8.0, 1 mM PMSF and 1:1,000 protease inhibitor cocktail and rotated at 4 °C for 2–4 h. The mixture was adjusted to 500 mM NaCl, allowed to rotate for another 45 min and then centrifuged at 13,500g for 10 min, yielding nucleosomes in the supernatant. Chromatin (40 μg per ChIP) was diluted to 100 ng/μl with Native buffer B (20 mM Tris, pH 8.0, 5 mM EDTA, 500 mM NaCl and 0.2% Tween 20). 1% was took as Input and the rest incubated overnight at 4 °C with specific antibodies. The chromatin was incubated for 3 h at 4 °C with 40 ul of ChIP-grade Magna ChIP protein A/G magnetic beads. The beads were washed three times with Native buffer B and once with Native buffer B without Tween. 100 ul of pK buffer (1x TE, 1mM CaCl2, 50mM NaCl, 0.5% SDS, 0.1ug/ul RNAse A) were added to the Input samples and the beads and incubated in a Thermomixer at 37°C for 1 hour, 2000 rpm. 3ul of Proteinase K were added and incubated for 3h at 56 °C, 2000 rpm. The DNA was purified using the MinElute PCR purification kit from Qiagen following the kit's instructions, eluted in 30ul of Qiagen's elution buffer and its concentration determined with an Agilent 2100 Bionanalyzer High Sensitivity Kit.  25ng of ChIPed or Input DNA were 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 2500 illumina platform.
attributes.tag: GEO Accession
attributes.value: GSM2066633