Pyromark assay design version 2

Genomic DNA was isolated from frozen hippocampal tissue by phenol-chloroform method [22 (link)]. Next, 500 ng of genomic DNA was bisulfite converted using the EpiTect Bisulfite Kit (Qiagen, Redwood City, CA, USA) according to the manufacturer’s protocol. Primers to amplify and sequence two promoter regions of PLD3 were designed with PyroMark Assay Design version 2.0.1.15 (Qiagen) (Additional file 1: Table S2), and PCR reactions were carried out on a VeritiTM Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Next, 20 μl of biotinylated PCR product was immobilized using streptavidin-coated sepharose beads (GE Healthcare Life Sciences, Piscataway, NJ, USA) and 0.3 μM sequencing primer was annealed to purified DNA strands. Pyrosequencing was performed using the PyroMark Gold Q96 reagents (Qiagen) on a PyroMark™ Q96 ID System (Qiagen). For each particular cytosine-phosphate-guanine dinucleotide (CpG), methylation levels were expressed as percentage of methylated cytosines over the sum of total cytosines. Unmethylated and methylated DNA samples (EpiTect PCR Control DNA Set, Qiagen) were used as controls for the pyrosequencing reaction.

Genomic DNA was isolated from frozen cell pellets of basal NPCs and control or Aβ peptide treated NPCs incubated in differentiation media for 9, 19, or 29 days by using the FlexiGene DNA Kit (Qiagen, Redwood City, CA, USA). Next, 500 ng of genomic DNA was bisulfite converted using the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s protocol. Primer pairs to amplify and sequence the chosen CpG genomic positions were designed with PyroMark Assay Design version 2.0.1.15 (Qiagen) (Supplementary Table S1) and bisulfite PCR reactions were carried out on a VeritiTM Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Next, 20 μL of the biotinylated PCR product was immobilized using streptavidin-coated Sepharose beads (GE Healthcare Life Sciences, Piscataway, NJ, USA) and 0.4 μM of sequencing primer annealed to purified DNA strands. Pyrosequencing was performed using PyroMark Gold Q96 reagents (Qiagen) on a PyroMark™ Q96 ID System (Qiagen). For each particular CpG, DNA methylation levels were expressed as the percentage of methylated cytosines over the sum of total cytosines. Unmethylated and methylated DNA samples (EpiTect PCR Control DNA Set, Qiagen) were used as controls for the pyrosequencing reaction.