title: GSM3100484: 24hr Baseline rep 3 [ChIP-seq]; Homo sapiens; ATAC-seq
library_construction_protocol: ATAC-seq was performed using the protocol outlined by Buenrostro et al. (2015). Briefly, cells were washed with 50uL cold 1x PBS buffer, then centrifuged at 500g for 5 minutes at 4°C. Cells were then resuspended in 50uL cold lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) , gently lysed to preserve cell nuclei, and centrifuged again at 500g for 10 minutes at 4°C. Cells were then washed 3x with wash buffer (lysis buffer without igepal). After each wash, cells were centrifuged at 500g for 5 minutes at 4°C. After washing, cell nuclei were resuspended in the transposition reaction mix and incubated for 30 minutes at 37°C. After transposition, DNA was purified using a Qiagen MinElute PCR Purification Kit and eluted in 10uL elution buffer. DNA was stored at -20°C until fragments were amplified via PCR. All of the DNA purified following transposition was PCR amplified. To do so, the following were combined in a 0.2mL PCR tube: 10uL transposed DNA, 10uL nuclease-free H2O, 2.5uL 25uM custom Nextera PCR primer 1, 2.5uL 25uM custom Nextera barcoded PCR primer 2, 25uL NEBNext high-fidelity 2x PCR master mix. The components were amplified as follows: 1 cycle of 72C for 5 min, 98C for 30 sec; 5 cycles of 98C for 10 sec, 63C for 30 sec, 72C for 1 min. After initial amplification, the number of additional cycles to run was determined using qPCR. For this, the following were combined in a 0.2mL PCR tube: 5uL of previously PCR-amplified DNA, 4.5uL of nuclease-free H2O, 0.25uL of 25uM custom Nextera PCR primer 1, 0.25uL of 25uM custom Nextera PCR primer 2, 5uL KAPA SYBR FAST qPCR master mix (2x) (total reaction 15 ul). The components were amplified as follows: 1 cycle of 98C for 30 sec; 20 cycles of 98C for 10 sec, 63C for 30 sec, 72C for 1 min. To calculate the number of additional cycles of PCR needed, linear Rn versus cycle was plotted and the cycle number that corresponds with 1/3 of the maximum fluorescent intensity was determined. After the number of additional PCR cycles was determined, the remaining 45uL of PCR product was run as follows: 1 cycle of 98C for 30 sec; N cycles (as determined via qPCR) of 98C for 10 sec 63C for 30 sec, 72C for 1 min. Finally, the amplified library was purified using 0.9x Ampure beads at room temperature, and eluted in 20uL RNase-free H2O.
attributes.tag: GEO Accession
attributes.value: GSM3100484