title: GSM3398627: 70neg; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Genomic DNA from each NeuN+ and NeuN- fraction of each sample was isolated using standard phenol-chloroform extraction methods. DNA methylation was captured using the SeqCap Epi Enrichment System (Roche). Biotinylated long oligonucleotide probes were customed designed by Roche NimbleGen. Library preparation was performed following manufacturer instructions. Briefly, gDNA (500 ng) of each sample (n= 4 cases and 4 controls, in addition 4 technical replicate samples) were fragmented (~ 200 bp), end repaired and ligated to barcoded adapters using the KAPA Library Preparation kit (Kapa Biosystems) and SeqCap Adapter Kit A and B (Roche). Bisulfite conversion of the adapter ligated DNA followed by column purification was performed with the EZ DNA Methylation Lightning kit (Zymo). The bisulfite converted DNA for each sample was then amplified by ligation mediated PCR (95°C 2 min, 10 cycles of [98°C 30 sec, 60°C 30 sec, 72°C 4 min], 72°C 10 min, 4°C hold) followed by purification with Agencourt AMPure XP beads (Beckman Coulter). Sample quality was verified on Bioanalyzer (Agilent) and quantity was determined with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Equimolar amounts of each sample were then combined into a single pool. Target regions were captured by hybridizing the amplified bisulfite converted DNA pool (1 μg) to the probe library (Roche), as directed by manufacturer. Enrichment and recovery of captured bisulfite-converted DNA was completed by binding to magnetic beads and subsequent wash steps using the SeqCap Pure Capture Bead kit and the SeqCap Hybridization and Wash kit (Roche). The captured DNA was then amplified by ligation mediated PCR (98°C 45 sec, 11 cycles of [98°C 15 sec, 60°C 30 sec, 72°C 30 sec], 72°C 1 min) followed by purification with Agencourt AMPure XP beads (Beckman Coulter). Library quality and quantity was assessed using a combination of Agilent DNA High Sensitivity chip on a Bioanalyzer (Agilent Technologies), Qubit dsDNA HS Assay kit on a Qubit 3.0 fluorometer (Thermo Fisher Scientific), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). DNA sequencing was performed on an Illumina HiSeq 2500with all samples run on both sequencing lanes.