|Public on Jun 19, 2020
braak stage: 6
tissue: Brain prefrontal cortex
molecule subtype: hMeDIP DNA
brain bank: Parkinson’s UK Brain Bank
replicate: Biological Sample
|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.
|Illumina NovaSeq 6000
|Brain bank Sample code:PD201
|library strategy: hMeDIP-seq
Adapter sequences were removed from fastq files using TrimGalore (v0.4.4).
Sequenced reads from hMeDIP immunoprecipitated and input control human brain nuclei were mapped to the human reference genome (GRCh37/hg19) with Bowtie2 (v2.3.1).
A combination of Picard and Samtools (v1.9) was used to mark and remove PCR duplicates respectively.
Deeptools (v2.3.1) was used to apply a Signal Extraction Scaling (SES) factor normalized to the input (1 kb wide tiling windows) prior to peak calling.
Broad peaks were called using MACS2 for each normalized sample and a consensus peak list was then defined as contiguous regions covered by at least 22 sample peaks. Consensus peaks in the shortest 5% and and longest 5% range were removed from further analysis.
To determine hydroxymethylation contribution to epigenetic changes at enhancers in PD neurons, hydroxymethylation counts for the differentially methylated cytosines in PD were extracted using bedtools coverage
Supplementary_files_format_and_content: Each bed file contains a bed 12 format for the macs2 broad peaks. Each txt file contains the following information in order: cytosine site location, read counts.
|Aug 05, 2019
|Last update date
|Jun 20, 2020
|616 920 9824
|Van Andel Research Institute
|Center for Neurodegenerative Science
|333 Bostwick Ave NE
|Epigenomic analysis of Parkinson’s disease neurons identifies TET2 loss as neuroprotective [PD_hMeDIP_Seq]
|Epigenomic analysis of Parkinson's disease neurons identifies TET2 loss as neuroprotective