title: GSM8507223: F-WT-Noco-ATAC-Seq-REP2; Mus musculus; ATAC-seq
library_construction_protocol: 50,000 of freshly harvested cells were resuspended in ice-cold RSB buffer (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl2). The suspension was centrifuged at 500xg for 3min using a refrigerated centrifuge. The cell pellet was washed once with 50μl ice-cold PBS and centrifuged at 500xg for 3min at 4°C. The cell pellet was resuspended in ice-cold Lysis Buffer (48.5μl RSB buffer supplemented with 0.5μl of 10% NP-40, 0.5μl of 10% Tween-20, 0.5μl of 1% Digitonin) and kept on ice for 3min. Then, 1ml of wash buffer (RSB buffer supplemented with 0.1% Tween-20) was added on the lysed cells. Nuclei were pelleted at 500g for 10min at 4°C and the supernatant (cytoplasm fraction) was carefully discarded. The nuclei were resuspended in the transposition mix (1x Tagment DNA Buffer (Illumina, #20034197), 0.33X PBS, 0.1% Tween-20, 0.01% Digitonin). Next, 2.5μl of Tn5 Transposase (Illumina, #20034197) was added to the reaction mix by pipetting up and down 6 times. The reaction was then incubated at 37°C for 30min on a thermomixer at 1000rpm. Finally, the tagmented DNA was isolated using Qiagen MinElute Reaction Cleanup-Kit (Qiagen, #28204) following the manufacturer's instructions. Nextera Index XT Kit v2 (FC-131-2001, Illumina)