GSM9289626: shDKD-CTRL-R1; Homo sapiens; ATAC-seq
NCI-H661 cells were harvested and counted after staining cells with 0.4% Trypan blue where 500,000 live cells were used for each ATAC assay. Cells were washed with ice cold PBS once and then lysed in 500uL NE buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630, 0.1% Tween-20) on ice by gentle pipetting to isolate nuclei. 50uL of nuclei (50,000 cells), were then pelleted at 1500xg for 10mins at 4C followed by supernatant aspiration. Nuclei were gently resuspended directly in tagmentation mix (1.75uL Tn5 transposase (Illumina), 17uL TD buffer (Illumina), 21.25uL H2O) on ice and then allowed to tagment for 37C for 30 mins in a thermalcycler. Tagmented DNA was then purified using Ampure XP beads at a 1.8X ratio and eluted in 21uL of H2O for library preparation as previously described using 15 cycles of PCR to incorporate i7/i5 indices followed by a final 1.8X Ampure XP bead cleanup and elution in 22uL of H2O.