title: GSM5350976: ATAC-seq WT rep 3; Mus musculus; ATAC-seq
library_construction_protocol: ATAC-seq was preformed using an adaptation of protocols described in (Buenrostro et al. 2016; King and Klose, 2017). In short, 100.000 cells were used per sample and centrifuged at 2000xg for 5 minutes at 4°C. Cells were washed with ice-cold 1x PBS and centrifuged at 2000xg for 5 minutes at 4°C. Nuclei were isolated using 50 μl Lysis buffer (10 mM Tris HCl ph 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40) and centrifuged immediately at 2000xg for 10 minutes at 4°C. Immediately after removing supernatant pellet was resuspended in 50 μl of TAPS-buffer (10 mM TAPS-NaOH pH 8.5, 5 mM MgCl2, 10% DMF) containing 25 nM adaptor-loaded, home-made Tn5E54K,L372P transposase (plasmids provided by Dr. Kim Remans, EMBL Heidelberg, Germany). The reaction was incubated for 1 hour at 37⁰C and was immediately purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Cat# 740609.50). DNA was eluted into a volume of 15 μl and stored at -20°C prior to library preparation. For library preparation, adaptors described in (Buenrostro et al. 2013; Buenrostro et al. 2015a) were used using Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific, Cat# ) with the following PCR program: 1 cycle of 5 minutes at 72°C for gap filling, 1 cycle of 30 seconds at 98°C for pre-denaturation, 14 amplification cycles with 10 seconds at 98°C for denaturation, 20 seconds at 72°C for primer annealing and extension and 5 minutes at 72°C for final extension. After purification using 1.5x SPRIselect beads (Beckman-Coulter, Villepinte, France, Cat# B23319), libraries were verified using capillary electrophoresis and subsequently sequenced with 2x 100 base pairs on a HiSeq4000 machine (Illumina). 