GSM4307491: P2D10_Bisulfite-seq_discovery; Homo sapiens; Bisulfite-Seq
Neuronal nuclei from prefrontal cotex were isoated by an antibody (NeuN) and flow cytometry approach; DNA were extracted by standard phenol chloroform extraction; Bisulfite padlock probes were designed to humans brain enhancers and promoters (NIH Roadmap Chromm) We scanned brain-relevant enhancers and promoters genome-wide using the bisulfite padlock probe approach. Padlock probes (n=59,009) were designed using the PP Designer software and synthesized using a programmable microfluidic microarray platform (CustomArray, Inc.). Isolated genomic DNA for each sample was bisulfite-converted and purified using the EZ DNA Methylation-Lightning Kit (Zymo Research). The bisulfite-converted DNA (200 ng) was hybridized to the padlock probes (1.5 ng). Targeted regions were extended using PfuTurbo Cx (Agilent Technologies) and circularization was completed using Ampligase (Epicentre). Non-circularized DNA was digested using an exonuclease cocktail and the remaining circularized DNA was amplified using a common linker sequence in the padlock probe. Libraries were PCR amplified, pooled in equimolar amounts, purified by QIAquick Gel Extraction kit (Qiagen) and quantified by qPCR (Kapa Biosystems) on a ViiA 7 Real-time PCR system (Applied Biosystems).