GSM2901720: Control Line 1 5iLAF 5% O2 SSEA4 Plus Reversion 1 [ATAC-Seq]; Homo sapiens; ATAC-seq
FACS collected cells were spun 500g x 5 minutes at 4°C. Cells were resuspended in 50μL lysis buffer (10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 1x Complete Protease Inhibitor(Roche)) and spun 10 minutes 500g 4°C to collect nuclei. The nuclei were resuspended in 50μL Transposase reaction (25μL 2xTagmentation buffer, 22.5μL water, 2.5μL Tn5 Transposase enzyme) and reacted for 30 minutes at 37°C on a PCR machine. The material was purified using a Qiagen MinElute protocol, eluting with 14μL EB. The eluted material was amplified in 50μL volume using 1.25μM primer concentration and a 1x concentration NEBNext High-Fidelity Master-Mix (NEB) (Program: 72°C 5:00, 98°C 30s, 5x (98°C 10s, 63°C 30s, 72°C 1 minute), 4°C hold). After these 5 cycles of amplification, the tube was kept on ice. A 5μL aliquot was then removed and used to perform a 15 μL side reaction with identical concentrations of primer and enzyme, except that 0.6x SYBR Green (Invitrogen S-7563) was included to monitor amplification. This side reaction was amplified on a Stratagene Mx3005p qPCR (Agilent) system with the following amplification conditions (98°C 30s, 20x (98°C 10s, 63°C 30s, 72°C 1:00)). The number of additional cycles 'N' required to reach ¼ maximum fluorescence was observed. The remaining 45μL of the reaction was then further amplified (98°C 30s, N x (98°C 10s, 63°C 30s, 72°C 1:00), 4°C) and the libraries were purified by a Qiagen MinElute kit eluting with 20 μL volume.
GEO Accession
GSM2901720