Persistent JunB Activation in Fibroblasts Disrupts Stem Cell Niche Interactions Enforcing Skin Aging [ATAC-seq]

ATAC-seq was performed in control murine dermal fibroblasts (MDF) in the absence and presence of rotenone at a concentration of 250µM for 3 hrs), and in freshly isolated control (Co) and Sod2 cKO MDF. Overall design: In brief, 50000 MDFs were lysed in Nuclei lysis buffer (10?mM Tris-HCl, pH 7.4, 10?mM NaCl, 3?mM MgCl2, 0.1% NP-40) for 2 min, followed by nuclei isolation using centrifugation (500 ´ g for 10 min at 4°C). Isolated nuclei were subjected to tagmentation reaction with Tn5 transposase (Illumina) for 30 min at 37°C. Tagmented DNA was then purified using MinElute kit (Qiagen) and used for PCR amplification to add the i5 and i7 dual barcodes. The PCR amplified products were purified by two times 1.8´ AMPure XP bead cleanup (Beckman Coulter). The purified library was then quality controlled by Qiaxcel advanced system (Qiagen) and Bioanalyzer (Agilent) and finally measured by Qubit fluorimeter (ThermoFisher Scientific). Thereafter, libraries were sequenced on Illumina NextSeq 500 platform (Microsynth GmbH) using NextSeq v2 chemistry (2 ´ 75 cycles).