title: GSM5654975: ATAC-seq_LPS_rep4; Mus musculus; ATAC-seq
library_construction_protocol: ATAC seq was performed as previously described (doi: 10.1038/nmeth.4396). In brief, 50.000 viable cells were lysed in 50µL resuspension buffer 1 (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40,0.1% Tween-20 and 0.01% Digitonin) on ice for 3 mins. The lysates were washed with resuspension buffer 2 ( 10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2,0.1% Tween-20). The nuclei were pelleted and resuspended in 50µL transposition mixture ( 25µL 2xTD buffer, 2.5µL Tn5 transposase (Illumina Tagment DNA enzyme and buffer kit #20034197), 16.5µL PBS, 0.5µL 1% digitonin, 0,5µL 10% Tween-20 and 5µL H2O). The reactions were incubated for 30 minutes at 37oC  agitating at 1000rpm and the transposed DNA was subsequently purified using the PCR MinElute purification kit (Qiagen). The tagmented DNA was initially amplified for 4 cycles using the NEBNext 2x PCR master mix (NEB) and Illumina compatible primers as described (https://doi.org/10.1038/nmeth.2688). The additional number of cycles for the amplification of the libraries were determined by a qPCR reaction using the amplified DNA as template and the Kappa SYBR Green (Roche) as indicated (doi: 10.1002/0471142727.mb2129s109). Libraries were further amplified and purified using the PCR MinElute kit (Qiagen). Size selection  was performed on purified libraries for fragments between 150-600bp the Agecount AMPure XP beads (Beckman Coulter)  and their quality was determined by the Qubit dsDNA HS kit (Thermo Scientific) and the Agilent High Sensitivity DNA kit on a  Bioanalyzer 2100.