GSM6069476: ATAC-seq biol. repl.2 DLD1-control; Homo sapiens; ATAC-seq
~50,000 cells form each replicate and conditions were washed in PBS and then re-suspended in nuclei lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). The crude nuclear pellet was re-suspended directly in Tn5 reaction mix (10 µl of 5X TTBL, µl TTE Mix V50, and 35 µl of RNase/DNase free H2O) using TruePrepTM DNA Library Prep Kit V2 for Illumina® TD501 from VAZYME). The reactions mix were then incubated at 37 oC for 30 min, and kept on ice. The DNA was purified in 20 µl of 10 mM Tris, pH8 using MiniElute KIT (Qiagen). DNA was amplified for an initial 5 cycles using the full eluate of transposed DNA, 2.5 µl of customized Nextera universal primer, 2.5 µl of customized Nextera barcoded primer and 25 µl of NEBNext High-Fidelity 2x PCR Master Mix (NEB). In the meantime, qPCR was set up in a total volume of 15 µl to determine the optimal PCR cycles for libraries amplifications. For each reaction, the number of PCR cycles that were added, were calculated from the plot of Rn vs. Cycle that corresponded to ¼ of maximum fluorescent intensity. Size selection of DNA was performed to eliminate the bulk of primer-dimer artifacts after libraries amplifications as described (PMID: 32913232). Namely,libraries were size selected by adding 1.3x volume of Agencourt AMPure XP magnetic beads (Beckman Coulter) to each PCR reaction, and eluted in 23 µl of 10 mM Tris, pH 8, after which, they  were quantified by Qubit using Quant-iT™ PicoGreen™ dsDNA Assay Kit, and sent for sequencing.  Libraries qualities were determined with The Agilent 2200 TapeStation system.