GSM6754083: 12a9_1614 (12 months, female); Mus musculus; Bisulfite-Seq
100,000 cerebral endothelial cells were sort-ed in FACS buffer (1X PBS, 2%FCS and 2mM EDTA) at 4°C and centrifuged (500g, 5 minutes at, 4°C). The supernatant was discarded, the cells were snap frozen in liquid nitrogen and stored at –80°C till further processing. For lysing the cells, 200 µL solution A (25mM EDTA, 75mM NaCl), 200 µL solution B (10 mM EDTA, 10 mM Tris-HCl, 1% SDS) and 10 µL Proteinase K (20 µg/µL) were added to the frozen cell pellet, followed by a brief vortex and incubation at 55°C. Phenol-chloroform-isoamyl (25:24:1) and chloroform-isoamyl (24:1) were used for liquid phase separation of the genomic DNA. Glycogen (20 µg/ µL), 0.1x volume (20 µL) of 3M Sodium Ace-tate and 2.5x volume (500 µL) of ice-cold 100% ethanol were added and incubated overnight at –20°C for precipitation of genomic DNA. The pellet of genomic DNA obtained was washed with 70% ethanol and dissolved in 40 µL pre-warmed 1x Tris-EDTA (TE buffer) at 45°C for 2 hours. DNA concentration was measured using a Qubit double stranded high sensitivity DNA assay kit (Life Technologies) according to the manufacturer's instructions. Restriction was performed on 26 µL DNA template using 1 µL HaeIII restriction enzyme (New England Biolabs) and 3 µL 10x Cutsmart buffer (New England Biolabs) at 37°C for 18 hours. A-Tailing was performed with 1 µl Klenow Fragment (3′ → 5′exo−, 5 U/µl, NEB) and 1 µl dATP (10 mM, NEB) at 37 °C for 30 min followed by enzyme inactivation at 75 °C for 20 min. Unique molecular identifier adapters (TruSeq Single Index Set B, Illumina) were ligated using 1 µl adapters (10 µM), 0.5 µl T4 Ligase (2,000 U/µl, NEB), 2 µl ATP (10 mM, NEB) and 1 µl Cutsmart buffer (10X, NEB)  at 16 °C for 18 hours, followed by enzyme inactivation 65°C for 20 minutes. Bisulfite conversion and subse-quent cleanup was performed using EZ-DNA Methylation Gold Kit (Zymo Research) according to the manufacturer's instructions and the bisulfite-converted genomic library was eluted in 24 µL nuclease-free water. The library was amplified by polymerase chain reaction using 0.6 µl each of primers (10 µM, Primer i5: AATGATACGGCGACCACCGAGATCTACAC, Primer i7: CAA-GCAGAAGACGGCATACGAGAT), 0.6 µl Hot Start Taq (5 U/µl, Qiagen), 3 µl Hotstar PCR Buffer (10X, Qiagen), 1.2 µl MgCl2 (25 mM) and 2 µl dNTPs (10 mM) in a 30 µl reaction with 95°C for 15 min, 20 cycles of 95°C for 40 s, 58°C for 1 minute, 72°C for 1 min, 72°C for 12 min and hold at 4°C. The amplified RRBS 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 RRBS. The RRBS library was analyzed with Bioanalyzer High-Sensitivity DNA Analysis kit (Agilent), and the concentration was determined using the Qubit HS DNA kit (Life Technologies). The sequencing was performed on Illumina HiSeq2500 sequencing system at the University of Saarland, Saarbrücken, and generated ap-prox. 50 Million 100 bp single-end reads per sample.ATAC-seq data processing. The adapter sequence and 3' ends with base quality (PHRED score) of less than 20 in the FASTQ files obtained from the sequencing were trimmed with Trim Galore (v0.4.2) software. Following adapter trimming and removing low quality nucleotides, the FASTQ files were mapped to the mouse reference genome (GRCm38/mm10)  using the GEM62 mapper. Duplicated reads found after alignment with GEM mapper were annotated with Picard tools (v1.115) (http://broadinstitute.github.io/picard). MACS263 (v2.1.0) was used to call Nucleosome Depleted Regions (NDRs) after downsampling the reads into similar number to avoid any bias in the downstream analyses due to sequencing depth. The parameters used in the MACS2 were: –shift -100, –extsize 200, –nomodel and –keep-dup all. Differentially accessible regions were calculated using a linear model accounting for gender. Peaks which overlap with the black list regions defined by ENCODE (https://doi.org/10.1038/s41598-019-45839-z) were excluded from the analysis. RRBS data processing. Sequencing reads were trimmed using the Trim Galore (v0.4.2) soft-ware (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove the adapter contamination and the 3' ends with base quality (PHRED score) of less than 20. The trimmed reads were then aligned to the mouse reference genome (GRCm38/mm10) using the BWA64 (v0.6.2) wrapper methylCtools65 (v0.9.2). Samtools66 (v1.3) and Picard tools (v1.115) (http://broadinstitute.github.io/picard) were used for converting, merging, and indexing of the alignment files. SNP aware realignment to identify single nucleotide polymorphisms (SNPs) for accurate identification of methylated cytosines and methylation calls were performed with the bisulfite SNP calling software Bis-SNP67. MethylKit68 (v1.3.1) was used for tiling (1kb tiles, min 3 CpGs per tile, each CpG with minimum coverage of 5) . Association of DNA methylation and ageing was calculated using a linear regression model with gender and coverage as covariates. Obtained p-values were corrected for multiple testing using Benjamini Hochberg correction. endothelial cells from brain were FACS enriched. RRBS protocol using restriction enzyme HaeIII DNA was fragmented with HaeII restriction enzyme. Fragments were end-repaired and A-tailed, followed by NGS adaptor (methylated) ligation using T4 ligase. Fragments were then bisulfite converted and amplified by PCR.