GSM5964755: IFNb rep3 (ATAC-seq_IFNb); Mus musculus; ATAC-seq
Cells were washed twice in 1 mL ice-cold PBS, by centrifugation at 1200 g for 5 min. Cells were resuspended in 1 mL cold nuclei isolation buffer (0.32 M sucrose; 3 mM CaCl2; 2mM Mg Acetate; 0.1 mM EDTA; 10 mM Tris.HCl pH 8.0; 0.6% NP-40; 1mM DTT fresh) and placed on ice for 5 minutes. Cells were centrifuged at 700g for 5 min at 4°C. Supernatant was removed, 500 µL cold nuclei isolation buffer was added and samples were kept on ice for 3 min to further increase the number of isolated nuclei. Cells were centrifuged at 700g for 5 min at 4°C. Supernatants were removed and pelleted nuclei were gently resuspended in 200 µL cold Nuclei Resuspension Buffer (NRB) (50 mM Tris.HCl pH 8.3; 40% Glycerol; 5 mM MgCl2; 0.1mM EDTA). 50.000 nuclei (5µl) were resuspended in 7.5µl H2O, 12.5 µl TD buffer and 5 µl Tn5 transposase (Illumina). Nuclei were incubated for 30 min at 37 °C, while mixing on a plate shaker at 600 rpm. DNA was purified with Qiagen MinElute columns according to the manufacturer's instructions and eluted in 13µl of nuclease-free H2O. 12.5 µl purified DNA from the previous step were mixed with 5 µl 2.5 µM I7 index primer, 5 µl 2.5 uM I5 index primer (dual indexing), 2.5 µl Evagreen and 25 µl, 2x Q5 PCR Master Mix, (NEB). An end point PCR was run as follows: 5 min 72 ºC, 1 min 98 ºC, 10sec 98 ºC (5-7 cycles) 30sec 65 ºC (5-7 cycles), 60sec 72 ºC (NEB). The reaction was purified by adding 15 µl of SPRI beads prepared by the NGS facility (MBSpure beads) and incubated for 5 min at RT. The supernatant was transferred to a fresh well. 50µl of MBSpure beads were added and mixed well, incubated for 5 min at RT, magnetized, and the supernatant was removed. The beads were washed twice by adding 150 µl of 80% EtOH. Beads were dried for 30sec and DNA was eluted in 20 µl of nuclease-free water. The quality of the libraries was checked on a bioanalyzer to further determine the size distribution. Libraries were sequenced on a NovaSeq SP PE50.