|
| Status |
Public on Jun 19, 2020 |
| Title |
PD6_A1_PD_TET2_Bisulfite_Padlock_Seq |
| Sample type |
SRA |
| |
|
| Source name |
PD_TET2_Bisulfite_Padlock_Seq
|
| Organism |
Homo sapiens |
| Characteristics |
Sex: M
age: 87
pmi: 19
braak stage: 6
tissue/cell type: Brain prefrontal cortex neurons(NeuN+)
brain bank: Parkinson’s UK Brain Bank
replicate: Biological Sample
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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.
Libraries were prepared using a targeted bisulfite sequencing approach known as bisulfite padlock probes sequencing. Padlock probes were designed to TET2 gene and surrounding area (±300 kb) and synthesized using a programmable microfluidic platform (Custom Array, Inc.). Genomic DNA was bisulfite converted and purified using the EZ DNA Methylation-Lightning Kit (Zymo Research). The bisulfite-converted DNA (200 ng) was then hybridized to the padlock probes (1.5 ng). PfuTurbo Cx (Agilent Technologies) was used to extend across the target regions 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). Next-generation sequencing of the libraries was performed on an Illumina HiSeq 2500 machine on HiOutput mode.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Brain bank Sample code:PD201
|
| Data processing |
Libraries sequenced on the HiSeq 2500 platform (Illumina) using custom sequencing primers.
Adapter sequences were removed from fastq files using TrimGalore (v0.4.4).
The remaining sequencing reads were then aligned to the target reference genome (GRCh37/hg19) indexed by Bismark (v0.17.0).
DNA methylation calls were counted using Bismark (v0.17.0).
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Each txt file contains the following information in order: chromosome #, genomic coordinate, strand, unmethylation count, methylation count, CHH/CHG/CG context of cytosine, exact sequence of the previous context
|
| |
|
| Submission date |
Aug 02, 2019 |
| Last update date |
Jun 20, 2020 |
| Contact name |
Lee Marshall |
| E-mail(s) |
lee.marshall@vai.org
|
| Phone |
616 920 9824
|
| Organization name |
Van Andel Research Institute
|
| Department |
Center for Neurodegenerative Science
|
| Lab |
Viviane Labrie
|
| Street address |
333 Bostwick Ave NE
|
| City |
Grand Rapids |
| State/province |
MI |
| ZIP/Postal code |
49503 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE135332 |
Epigenomic analysis of Parkinson’s disease neurons identifies TET2 loss as neuroprotective [PD_TET2_Bisulfite_Padlock_Seq] |
| GSE136010 |
Epigenomic analysis of Parkinson's disease neurons identifies TET2 loss as neuroprotective |
|
| Relations |
| BioSample |
SAMN12477158 |
