title: GSM3732619: siE1A_ATACseq; Homo sapiens; ATAC-seq
library_construction_protocol: ATAC-seq (Omni-ATAC protocol) was performed as described in Corces et al. 2017. HEK293 cells siRNA transfected for 4 days were lifted from plates using Accutase (Innovative Cell Technologies, Inc.) and 50,000 viable cells were gently pelleted at 500 RCF 4°C for 5min and resuspended in 50 uL cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin. Cells were incubated on ice for 3min then washed with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 to lyse plasma membranes. Nuclei were gently pelleted at 500 RCF for 10min at 4°C and resuspended in 50 uL of transposition mixture: 25 uL 2x TD buffer and 2.5 uL transposase (both supplied in the Nextera DNA Library Prep Kit), 16.5 uL PBS, 0.5 uL 1% digitonin, 0.5 uL 10% Tween-20, 5 uL H2O. DNA was isolated using DNA Clean and Concentrator-5 Kit (Zymo cat# D4014) and eluted in 21uL of elution buffer. 20uL of eluted DNA was amplified for 5 cycles Amplify for 5 cycles using NEBNext 2x MasterMix with 25uM primer Ad1 + 25uM primer Ad2 or 25uM Ad1 + 25uM Ad3. Refer to Corces et al. 2017 for PCR protocol and primer sequences. 5uL of preamplified sample was used in qPCR to determine additional cycles needed.
attributes.tag: GEO Accession
attributes.value: GSM3732619