GSM6754073: 13a3 (12 months, male); Mus musculus; ATAC-seq
Cerebral endothelial cells were sorted into FACS buffer (1X PBS, 2%FCS and 2mM EDTA) at 4°C and the assay for transposa-se-accessible chromatin sequencing (ATAC-seq) was performed according to a protocol adapted from Buenrostro et al57. 50,000 endothelial cells were spun down (500g, 5 minutes, 4°C), supernatant was removed and the cell pellet was resuspended in cold DNase inhibiting buffer (1 M KCl, 5 M NaCl, 1 M Tris-HCl, 0.5 M EGTA and 0.5 M Spermidine) containing prote-ase inhibitor cocktail (Roche). 0.1% IGEPAL® CA-630 (Sigma) was added to the suspension followed by gently inverting the tube 3-4 times and incubation on ice for 5 minutes. The nuclei were spun down (500g, 5 minutes, 4°C), supernatant was removed and resuspended in the DNase inhibiting buffer. The nuclei were again centrifuged (500g, 9 minutes, 4°C), the superna-tant was removed, and the pellet was resuspended in 50 µL reaction mixture, containing 25 µL 2X TD buffer, 22.5 µL nuclease-free water, and 2.5 µL Tn5 transposase enzyme (Nextera DNA Library Preparation Kit, Illumina, FC-121-1030). The tagmentation reaction was performed at 37 °C for 30 min followed by purification of library using MinElute PCR Purification Kit (Qiagen). The library was eluted in 26 µL elution buffer (Qiagen) and stored at –80°C until amplification. For amplification, 20 µL library was added to 30 µL PCR mix containing 25 µL NEB Next High Fidelity 2X Master Mix (New England Biolabs), 1 µL each Nextera i5 and i7 indexed primers as forward and reverse primers, and 3 µL nuclease-free water. The amplification was carried out in following steps – one cycle of 72°C for 5 minutes and 98°C for 30 seconds, 12 cycles of 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute and one cycle of 72°C for 5 minutes. The amplified library was cleaned using 0.8x volume (40 µL) of Ampure beads XP (Beckman Coulter) according to the manufacturer's instruction and eluted in 20 µL of 0.1X TE buffer. Six animals (3 males and 3 females) belonging to each of the age-groups of 2, 6, 12 and 18 months (total n = 24) were used for the ATAC-seq study. The quality of the ATAC library was analyzed with Bioanalyzer High-Sensitivity DNA Analysis kit (Agilent) and the concentration of the library was determined using the Qubit HS DNA kit (Life Technologies). The ATAC libraries were sequenced on an Illumina HiSeq2500 sequencing system at the University of Saarland, Saarbrücken and generated approx. 50 million 100 bp paired end reads for each sample. endothelial cells from brain were FACS enriched. native nuclei were tagmented with Tn5 transposase which was preloaded with Nextera NGS adaptors. After column-based purification, fragmented were amplified by PCR.