title: GSM1665990: SKmel147_Input_control; Homo sapiens; MNase-Seq
library_construction_protocol: Chromatin from SK-mel147 cells and eGFP-H2A, eGFP-H2A.Z.1 or eGFP-H2A.Z.2-transduced cells was digested with micrococcal nuclease (MNase) and used for ChIP with H2A.Z and GFP Trap Beads (Chromotek), essentially as described (Hasson et al., 2013). Sequencing libraries were generated and barcoded for multiplexing as previously described (Hasson et al., 2013) and resulting libraries were submitted for 100-bp, single-end Illumina sequencing on a HiSeq 2000 instrument. Total of 25ng MNase-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.
attributes.tag: GEO Accession
attributes.value: GSM1665990