GSM4746069: 22RV1_DMSO_ATAC_1; Homo sapiens; RNA-Seq
50,000 live cells in culture medium were treated with DNase at a final concentration of 200 U/ml at 37C for 30 minutes. The cells were washed thrice with PBS to remove DNase followed by lysis with RSB (10mM Tris-HCl, 10mM NaCl, 3 mM MgCl2) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and mixed by pipetting up and down 3 times. The cells were lysed on ice for 3 minutes and the reaction was stopped by adding 1ml of ice cold RSB containing 0.1% Tween-20 and mixed by inverting the tubes thrice. The nuclei were pelleted by centrifugation at 4C and the pellet was resuspended in 50ul of transposition mix (25ul 2x TD buffer, 2.5ul transposase, 16.5ul PBS, 0.5ul 1% digitonin, 0.5ul 10% Tween-20, 5ul H2O) and was incubated at 37C for 30 minutes with mixing. The reaction was then cleaned up with a Zymo DNA Clean and Concentrator-5 Kit. Library preparations for the samples were performed as described in Buenrostro et al., 2013 with minor changes. In brief to generate multiplexed libraries, A preamplification cycle was performed with the transposed DNA. The DNA was amplified for 5 PCR cycles with Illumina indexed primers (Nextera index kit, Illumina), 25ul of NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs). 5ul of this amplified DNA was amplified further using a qPCR to determine the appropriate number of cycles of PCR cycles needed to achieve maximum amplification with minimum duplicates. The number of cycles needed was calculated by finding the cycle number that corresponds to 1/4th of the maximum fluorescence intensity. The final amplification was performed in the leftover 45ul from the preamplification reaction using the number of cycles calculated in the qPCR.