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| Status |
Public on May 03, 2019 |
| Title |
75pos |
| Sample type |
SRA |
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| Source name |
frontal cortex of the human brain
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| Organism |
Homo sapiens |
| Characteristics |
project.sampleid: 75
tissuebank.id: HCcFH
age: 35
Sex: Male
race: White
dist.dx: Control
tissuebank: UM Brain Endowment Bank
tissue: brain frontal cortex
cell type: NeuN+
pmi: 12
molecule subtype: Bisulfite converted DNA from NeuN+ cells
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| 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.
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| Growth protocol |
DNA was extracted from post-mortem brain samples. No growth protocols were used.
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| 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.
DNA methylation was captured using the SeqCap Epi Enrichment System (Roche). Biotinylated long oligonucleotide probes were customed designed by Roche NimbleGen. Library preparation was performed following manufacturer instructions. Briefly, gDNA (500 ng) of each sample (n= 4 cases and 4 controls, in addition 4 technical replicate samples) were fragmented (~ 200 bp), end repaired and ligated to barcoded adapters using the KAPA Library Preparation kit (Kapa Biosystems) and SeqCap Adapter Kit A and B (Roche). Bisulfite conversion of the adapter ligated DNA followed by column purification was performed with the EZ DNA Methylation Lightning kit (Zymo). The bisulfite converted DNA for each sample was then amplified by ligation mediated PCR (95°C 2 min, 10 cycles of [98°C 30 sec, 60°C 30 sec, 72°C 4 min], 72°C 10 min, 4°C hold) followed by purification with Agencourt AMPure XP beads (Beckman Coulter). Sample quality was verified on Bioanalyzer (Agilent) and quantity was determined with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Equimolar amounts of each sample were then combined into a single pool. Target regions were captured by hybridizing the amplified bisulfite converted DNA pool (1 μg) to the probe library (Roche), as directed by manufacturer. Enrichment and recovery of captured bisulfite-converted DNA was completed by binding to magnetic beads and subsequent wash steps using the SeqCap Pure Capture Bead kit and the SeqCap Hybridization and Wash kit (Roche). The captured DNA was then amplified by ligation mediated PCR (98°C 45 sec, 11 cycles of [98°C 15 sec, 60°C 30 sec, 72°C 30 sec], 72°C 1 min) followed by purification with Agencourt AMPure XP beads (Beckman Coulter). Library quality and quantity was assessed using a combination of Agilent DNA High Sensitivity chip on a Bioanalyzer (Agilent Technologies), Qubit dsDNA HS Assay kit on a Qubit 3.0 fluorometer (Thermo Fisher Scientific), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). DNA sequencing was performed on an Illumina HiSeq 2500with all samples run on both sequencing lanes.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Trimmomatic v0.36 was used to trim read adapter sequences and BSMAP 2.74 was used to align reads to the GRCh38/hg38 genome build. The genome index consisted of reference chromosome sequences and the lambda phage genome (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/nuccore/215104).
Reads was separated into those aligning to top and bottom strand, duplicates were removed with Picard (2.9.4 SNAPSHOT) , and then matching read pairs were merged.
Samtools v1.5 was used to exclude reads that were not properly paired or were unmapped. Bamutils were used to clip overhanging reads that distort methylation estimates. Methratio.py in BSMAP computed the percent methylation at the base level.
Genome_build: hg38
Supplementary_files_format_and_content: Tab-delimited file with base-level calls of methylated/unmethylated reads at a Cytosine. Output of methratio.py in BSMap software
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| Submission date |
Sep 24, 2018 |
| Last update date |
May 03, 2019 |
| Contact name |
Shraddha Pai |
| E-mail(s) |
shraddha.pai@utoronto.ca
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| Organization name |
University of Toronto
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| Street address |
160 College Street, Room 602
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| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE112524 |
DNA methylation in neurons from post-mortem brains in schizophrenia and bipolar disorder (Bisulfite-Seq) |
| GSE112525 |
DNA methylation in neurons from post-mortem brains in schizophrenia and bipolar disorder |
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| Relations |
| BioSample |
SAMN10107979 |
| SRA |
SRX4731896 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3398632_75pos.methylation_results.txt.gz |
169.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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