GSM8775418: G7883_treated_parental_H226_cells_rep1 (Micro-Capture C); Homo sapiens; OTHER
Cells were scraped and crosslinked in the presence of 1% formaldehyde (in PBS) for 10-minutes in suspension followed by the addition of 1.5ml 1M Glycine and further incubation for 5-minutes at room temperature. Cells were washed in ice-cold PBS, counted, and 3-4x106 cell aliquots were prepared for nuclei isolation with the following MCC lysis buffer: 10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% Digitonin and 1% BSA in nuclease-free H2O. Each cell aliquot of 3x106 was resuspended in 500ul MCC lysis buffer and incubated on ice for 5-minutes. Cells were spun down, washed in ice-cold PBS, and washed in nuclease-free H2O before proceeding to MNase digestion with 90-130U of MNase for 3x106 cells that went on for 1-hour at 37C while shaking in a thermomixer (Eppendorf) at 550rcf. MNase was stopped by the addition of EGTA. At this point, nuclei were split into two tubes, and end-repair and proximity ligation were performed overnight. On the next day, nuclei were spun and subjected to proteinase K digestion at 65C overnight. DNA was column purified (Minelute, Qiagen) and eluted in 130ul H2O for sonication with Covaris E220 model using the following settings: Fill Level – 10, Duty Cycle – 15, PIP – 300, Cycles/Bursts – 200; sonicated for 5-minutes. DNA was cleaned up with Minelute, quantified and subjected to library preparation with Ovation Ultralow V2 Library Systems (Tecan) using 100ng DNA and 8 PCR cycles. Amplified libraries were subjected to hybridization with biotinylated oligonucleotide pools that were designed by the HyperDesign Team (Roche) and synthesized by Roche. All hybridization reagents were purchased from Roche as part of the KAPA Hyper Prep kit, and hybridization, pull-down, and washes were performed per manufacturer's instructions. Bead-bound libraries were amplified with the PCR reagents from the Ovation Ultralow Library V2 Kit (Tecan). Libraries were quantified and sequenced on a NovaSeq 6000 with 2x150bp configuration. MCC analysis was performed on two biological replicates per condition, and each biological sample was defined as a pooled total of 3 different MNase conditions (90U, 110U and 130U, 3x106 cells per MNase reaction), each split into two ligation reactions for subsequent downstream steps as described above. OTHER: Micro-Capture C (MCC)