Streptavidin sepharose hp beads

The PCR program consisted of a denaturing step of 3 min at 95°C followed by 45 cycles of 15 s at 95°C, 20 s at 54°Cand 30 s at 72°C, with a final extension of 5 min at 72°C.
The pyrosequencing samples were prepared using the Vacuum Prep workstation (Biotage AB, Uppsala, Sweden). The biotinylated amplicons were immobilized onto streptavidin Sepharose beads in the following reaction system:40 μl of the amplicon, 3 μl of streptavidin Sepharose HP beads (GE Healthcare), 37 μl of binding buffer [10 mM Tris-HCl, 2 M NaCl, 1 mM EDTA, 0.1% Tween-20, and Milli-Q (18.2 MΩ x cm) water,pH 7.6] and 15 μl of Milli-Q water. After one denaturation step and two washing steps using the Vacuum Prep workstation, the amplicons were transferred to a plate containing 0.4 μM sequencing primer in 40 μl of annealing buffer (20 mmol/l Tris-acetate and 2 mM magnesium acetate, pH 7.6). Pyrosequencing was performed using the PyroMark Gold Q96 Reagent and the PyroMark ID system (Qiagen, Germany), and sequences were analyzed with the Pyro Q-CpG™ software v. 1.0.9.PCR and sequencing primers were synthesized based on previously reported sequences[29 (link),30 (link)].

Targeted DNA methylation analysis, based on pyrosequencing, was performed in collaboration with a specialized service company (Varionostic, Ulm, Germany). For pyrosequencing analysis, samples were prepared according to standard procedures using a Vacuum Prep Tool. 40–50 μl PCR product was immobilized to 3 μl Streptavidin Sepharose HP beads (GE Healthcare, Chicago, IL, USA) followed by annealing to 2 μl sequencing primer (5 μM) for 2 min at 80 °C. For more information on primer sequences and assays used see Supplementary Tables 7 and 8. Analyses of CpGs were performed using Pyro Q-CpG software (Biotage, Uppsala, Sweden). Bisulfite conversion with subsequent pyrosequencing did not distinguish methylated and hydroxymethylated cytosines (5 mC vs 5 hmC, respectively).^{34 (link)}

Pyrosequencing of the three age-associated CpGs was measured in 243 blood samples of healthy controls and 105 [17 (link)] and 80 [25 (link)] of these were also mentioned in previous work. Furthermore, we analyzed 70 independent AA and 18 independent DKC patients. Pyrosequencing was described in detail before [17 (link)] and performed at Cygenia GmbH (Aachen, Germany). In short, DNA was isolated from patient samples with the QIAamp DNA Mini and Blood Mini kit (Qiagen, Hilden, Germany) and bisulfite converted with the EZ DNA Methylation Kit (Zymo Research, Freiburg, Germany). Converted DNA was PCR amplified with the PyroMark PCR kit 800 (Qiagen) and 20 μL PCR product was immobilized to 60 μL Streptavidin Sepharose HP beads (GE Healthcare, Chicago, IL, USA), annealed to 0.8 μL 20 μM sequencing primers for 2 min at 80 °C (see all primer sequences in Additional file 1: Table S4). Samples were measured with the PyroMark Gold Q96 Reagents on a PyroMark Q96 ID and analyzed with the Pyro-Q-CpG 1.0.9 software (all from Qiagen). Epigenetic age was calculated as follows:
Predicted age (in years) = 38.0 − 26.4 × β value (cg02228185) − 23.7 × β value (cg25809905) + 164.7 × β value (CpG upstream of cg17861230)

Sodium bisulfite-treated DNA was used to perform PCR to enrich the DNA template for pyrosequencing (Colella et al., 2003 (link)). Pyrosequencing was carried out using the PyroMask^® Gold Q24 machine (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Pyrosequencing primers were subsequently designed focusing on 3-5 targets CpG dinucleotides of TRDP, C16ORF89, ATAT1, and MSN. All primer sequences are depicted in Supplementary Table 1. Each sample was analyzed in triplicate for this experiment. In brief, 20-µl aliquots of the PCR products were immobilized on streptavidin Sepharose HP beads (GE Healthcare, Little Chalfont, UK) and purified using a PyroMark Q24 vacuum workstation (Qiagen) according to the manufacturer’s instructions. Then, the samples were denatured at 80°C for 2 min and annealed to a sequencing primer. Thereafter, the samples were subjected to analysis in a pyrosequencing machine. Nucleotide dispensation order and sequence analyses were performed with PyroMark Q24 software 2.0.6 (Qiagen). The pyrograms were analyzed in CpG assay mode to quantify the methylation percentage of each CpG.