|
| Status |
Public on May 03, 2019 |
| Title |
Sample 31 |
| Sample type |
genomic |
| |
|
| Source name |
FACS-sorted neurons from the frontal cortex of the human brain
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: frontal cortex of the brain
sampleID: 34
tissuebank.id: AN08747
age: 37
Sex: Male
race: White
dist.dx: Control
tissuebank: Harvard HBTRC
pmi: 18.75
cell.type: NeuN+
|
| Treatment protocol |
Neuronal nuclei were separated using a flow cytometry-based approach, similar to as previously described (PMID: 25369517,PMID: 19078943). Human brain tissue (250 mg) for each sample was minced in 2 mL PBSTA (0.3 M sucrose, 1X phosphate buffered saline (PBS), 0.1% Triton X-100). Samples were then homogenized in PreCellys CKMix tubes with a Minilys (Bertin Instruments) set at 3,000 rpm for three 5 sec intervals, 5 min on ice between intervals. Samples homogenates were filtered through Miracloth (EMD Millipore), followed by a rinse with an additional 2 mL of PBSTA. Samples were then place on a sucrose cushion (1.4 M sucrose) and nuclei were pelleted by centrifugation at 4,000 × g for 30 min 4°C using a swinging bucket rotor. For each sample, the supernatant was removed and the pellet was incubated in 700 μl of 1X PBS on ice for 20 min. The nuclei were then gently resuspended and blocking mix (100 μl of 1X PBS with 0.5% BSA (Thermo Fisher Scientific) and 10% normal goat serum (Gibco) was added to each sample. NeuN-488 (1:500; Abcam) was added and samples were incubated 45 min at 4°C with gentle mixing. Immediately prior to flow cytometry sorting, nuclei were stained with 7-AAD (Thermo Fisher Scientific) and passed through a 30 μM filter (SystemX). Nuclei positive for 7-AAD and either NeuN+ (neuronal) or NeuN- (non-neuronal) were sorted using an Influx (BD Biosciences) at the Faculty of Medicine Flow Cytometry Facility at the University of Toronto (Toronto, ON, Canada). Approximately 1 million NeuN+ nuclei were sorted for each sample. Immediately, after sorting nuclei were placed on ice and then precipitated by raising the volume to 10 mL with 1X PBS and adding 2 mL 1.8 M sucrose, 50 μl 1M CaCl2 and 30 μl Mg(Ace)2 and centrifugation at 1,786 × g for 15 min at 4°C. The supernatant was removed from NeuN+ and NeuN- samples and pellets were stored at -80°C.
|
| Growth protocol |
DNA was extracted from post-mortem brain samples. No growth protocols were used.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA from each NeuN+ and NeuN- fraction of each sample was isolated using standard phenol-chloroform extraction methods.
|
| Label |
Cy5 and Cy3
|
| Label protocol |
Reagent/array were purchased from Illumina. The experiment was carried out in Sickkids TCAG Microarray facility according to the recommended protocol by Illumina.
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| |
|
| Hybridization protocol |
Reagent/array were purchased from Illumina. The experiment was carried out in Sickkids TCAG Microarray facility according to the recommended protocol by Illumina.
|
| Scan protocol |
Reagent/array were purchased from Illumina. The experiment was carried out in Sickkids TCAG Microarray facility according to the recommended protocol by Illumina.
|
| Description |
neurons from post-mortem human brain
|
| Data processing |
Data generated from the microarrays were preprocessed with Minfi v1.19.12. Normalization was performed with noob, followed by quantile normalization. All samples had sex matching that predicted from the methylome. Probes that overlapping SNPs (MAF > 5%) on the CpG or single-base extension were excluded (11,812 probes), as were probes known to be cross-reactive (42,558 probes; PMID: 27330998) and those that failed detectability (P>0.01) in >20% samples (1,170 probes). After processing, 812,663 probes were left. Based on principal component analysis (PCA) co-clustering, one sample, despite being labeled NeuN+, clustered with NeuN- samples and was excluded from downstream analyses. Downstream analyses were performed using beta values ; only samples that were also genotyped were used in differential methylation analyses.
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| |
|
| Submission date |
Mar 21, 2018 |
| Last update date |
May 03, 2019 |
| Contact name |
Shraddha Pai |
| E-mail(s) |
shraddha.pai@utoronto.ca
|
| Organization name |
University of Toronto
|
| Street address |
160 College Street, Room 602
|
| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL21145 |
| Series (2) |
| GSE112179 |
DNA methylation in neurons from post-mortem brains in schizophrenia and bipolar disorder (Methylation) |
| GSE112525 |
DNA methylation in neurons from post-mortem brains in schizophrenia and bipolar disorder |
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