title: GSM1550784: E16.5 Morc1 het ATAC-seq Replicate 1; Mus musculus; OTHER
library_construction_protocol: FACS collected cells from individual mice 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 5 μL 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) is 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 1:100)). The number of additional cycles ‘N’ required to reach ¼ maximum fluorescence was observed. The purpose of this side reaction was to minimize the number of PCR cycles required used to generate the libraries, since length and GC bias increases with more amplification 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 puried by a Qiagen MinElute kit eluting with 20 μL volume