GSM4007975: PD6_17input_PD_hMeDIP_Seq; Homo sapiens; MeDIP-Seq
Neuronal nuclei from prefrontal cortex were isolated using an antibody (NeuN) and flow cytometry approach. Human prefrontal cortex brain tissue was homogenized and then filtered through Miracloth before being passed through a 1.4 M sucrose cushion by centrifugation at 4000 × g for 30 min 4°C. Nuclei pellets were resuspended and blocking mix (100 μl of 1X PBS with 0.5% BSA and 10% normal goat serum) was added to each sample. Anti-NeuN antibody with Alex Fluor 488 (1:500; Abcam; ab190195) was added and samples were incubated 45 min at 4°C with gentle mixing, followed by staining with 7-AAD. Nuclei positive for 7-AAD and either NeuN+ (neuronal) or NeuN‒ (non-neuronal) were sorted by flow cytometry. Genomic DNA from NeuN+ nuclei was extracted by standard phenol-chloroform extraction. Genomic DNA from human prefrontal cortex neuronal nuclei was sheared using a Covaris LE220 sonicator (size range of 200–600 bp) and hMeDIP was performed using the EpiQuik kit (Epigentek). 5-hmC antibody or non-immune IgG was coated on the bottom of a 96-well plate, wells were washed and either 1 µg sheared DNA, 1 µg control DNA, or buffer only was added to appropriate wells. The plate was then covered and incubated for 90 min with orbital rotation at 100 rpm. Wells were then washed 5 times with washing buffer. Captured DNA was then eluted by incubation of each well with a proteinase K mixture at 60°C for 15 min followed by incubation at 95°C for 3 min. The solution containing eluted DNA was transferred to another 96-well plate. DNA in each well was then purified using AMPure XP beads (Beckman Coulter). Libraries were prepared from 10 ng of input material or all available immunoprecipitated material using the KAPA Hyper Prep Kit (v5.16) (Kapa Biosystems). End repaired and A-tailed DNA fragments were ligated to IDT for Illumina TruSeq UD Indexed Adapters (Illumina), followed by PCR amplification. Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies), and QuantiFluor® dsDNA System (Promega Corp.). Sequencing (single-end 100 bp) was performed on an Illumina NovaSeq6000 sequencer.