GSM8723678: breast cells, E50K, Biological rep3; Homo sapiens; ATAC-seq
Cells were counted and 50,000 cells were washed with cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water), and permeabilized with ATAC-seq lysis buffer (RSB supplemented with 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin). For the transposition step, cells were resuspended in 44 µl of transposition mix (25 µl 2× TD buffer, 2.5 µl transposase, 16.5 µl PBS, 0.5 µl 1% digitonin, and 0.5 µl 10% Tween-20) and incubated at 37 °C for 40 min in a thermomixer with shaking at 1,000 RPM. The DNA fragments were cleaned up with the DNA Clean & Concentrator kit (Zymo Research, D4014) and PCR amplified for 8-12 cycles with Illumina Nextera adaptors using Kapa SYBR® FAST (Roche, 07959427001). The number of PCR cycles was determined based on monitoring for a change in fluorescence of 100,000 or a ΔRn of 0.1, after which PCR was stopped and the samples removed. After amplification, a second cleanup with 1.8x Ampure XP beads (Aline biosciences, Cat. C-1003-50) was performed and library eluted in 10mM Tris-HCl. Fragment size distribution and concentration were evaluated via Bioanalyzer High Sensitivity DNA kit (Agilent, 5067-4626), and libraries were sequenced to a target depth of at least 75 million paired-end reads per sample (Novogene).