title: GSM2066632: LAN6-ATRX-XL-MNase_ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: XL-MNase ChIP: Cross-linked MNase ChIP was performed using the Cell Signaling SimpleChIP Enzymatic Chromatin IP Kit with Magnetic beads (cat. #9003). The vendor's instructions were followed with some changes. Briefly: 20 million LAN6 cells were cross-linked for 10 minutes at room temperature in complete media, 100mM NaCl, 1mM EDTA pH 8.0, 50mM HEPES pH 8.0, 1% formaldehyde. Glycine was added to a final concentration of 125mM to stop the cross-linked reaction. The cells were washed once with cold PBS. Nuclei were obtained lysing the cells with 4ml of buffer A + DTT + Protease Inhibitor Cocktail (PIC) for 10 minutes at 4°C. Nuclei were washed with 3 ml of ice-cold buffer B + DTT. Nuclei were resuspended in 1ml of ice-cold buffer B + DTT, and digested with 8,000 gel U of Micrococal Nuclease from NEB (cat. #M0247S) at 37°C for 20 minutes. The reaction was stopped adding EDTA for a final concentration of 50nM and incubating on ice for 5 minutes. The nuclei were pelleted, and resuspended in 1ml of ChIP buffer + PIC + PMSF and incubated on ice for 10 min. The nuclei were split in two tubes and lysed by light sonication in a Bioruptor Twin (4 cycles, 20 sec ON/OFF, high power). The chromatin was cleared by centrifugation and a sample taken and processed (see bellow) to measure the concentration of the chromatin. 40ug of chromatin were diluted to 100ng/ul with ChIP buffer. 1% of the diluted chromatin was took as Input and the rest incubated with specific antibodies overnight at 4°C. 40 ul of ChIP-grade Magna ChIP protein A/G magnetic beads from Millipore (cat. #16-663) were added and incubated for 3 hours at 4°C. The beads were washed 3 times with 1ml of ChIP buffer, 1 time with 1ml of ChIP buffer + 350 mM NaCl, 1 time with 1 ml of LiCl buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 1% sodium deoxycholate, 1% Igepal, 250mM LiCl, not included in the kit) and 1 time with 1ml of TE. The beads and the Input samples were incubated in 100ul of Elution buffer for 30 min in a thermomixer at 65°C, 1200 rpm. The eluted material was incubated for 1 hour at 37°C 1ul of RNAse A (10mg/ml), followed with a 3 hour incubation at 55°C with 3ul of Proteinase K (20mg/ml). The cross-linked was reversed incubating the eluted material for 4-6 hours at 65°C. The DNA was purified using the MinElute PCR purification kit from Qiagen following the kit's instructions and eluted in 30ul of Qiagen's elution buffer. The amount and the pattern of the obtained DNA was determined using 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 NextSeq 500 illumina platform.
attributes.tag: GEO Accession
attributes.value: GSM2066632