Pyromark q96 md pyrosequencer

Two sets of bisulfite polymerase chain reaction (PCR) primers were designed to target each of two regions in the second intron of the human FKBP5 gene. The two regions flanked a glucocorticoid response element (GRE) [15 (link)] and contained consecutive cytosine-guanine dinucleotides (CpGs) implicated in previous studies [8 (link), 11 (link)]. The coordinates of the nine CpGs on chromosome 6 are Chr6: 35,606,441–35,606,662 (CpG1-4) and Chr6: 35,609,542–35,609,666 (CpG5-9), according to the UCSC Genome Browser build GRCh37.hg19 assembly [16 (link)]. Two rounds of a nested PCR amplification were performed. In the first PCR, 3.5 μL bisulfate converted DNA was used. In the second PCR, 2 μL template from the PCR was used. One of the nested bisulfite primers was biotinylated and HPLC-purified, allowing it to bind to sepharose beads and become single-stranded, in preparation for bisulfite pyrosequencing. The single-stranded amplicons were annealed to pyrosequencing primers and subjected to primer extension and nucleotide incorporation using the PyroMark Q96 MD pyrosequencer (Qiagen). The pyrosequencer QCpG program determined percent DNA methylation (DNAm) at all of the CpG dinucleotides downstream of the annealed primer at > 90% precision.

DNA from the same samples as above were subjected to bisulfite conversion using the Zymo EZ DNA Methylation Kit (Zymo Research, Irvine, California), which converts DNA methylation information into sequence base differences by deaminating unmethylated cytosines to uracil while leaving methylated cytosines unchanged. Bisulfite pyrosequencing assays were designed with PyroMark Assay Design 2.0 (Qiagen, Hilden, Germany; Supplementary Table 4). The regions of interest were amplified by PCR using the HotstarTaq DNA polymerase kit (Qiagen, Hilden, Germany) as follows: 15 min at 95°C, 45 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and a 5 min 72°C final extension step. For pyrosequencing, single-stranded DNA was prepared from the PCR product with the Pyromark™ Vacuum Prep Workstation (Qiagen, Hilden, Germany) and the sequencing was performed using sequencing primers on a Pyromark™ Q96 MD pyrosequencer (Qiagen, Hilden, Germany). The quantitative levels of methylation for each CpG dinucleotide were calculated with Pyro Q-CpG software (Qiagen, Hilden, Germany). Of note, only PAE and C animals were assessed by bisulfite pyrosequencing. We selected several DMRs for verification by bisulfite pyrosequencing based on their potential role in PAE-induced deficits, mainly focusing on their associated gene.