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Sample GSM2478426 Query DataSets for GSM2478426
Status Public on Dec 20, 2017
Sample type SRA
Source name acutely isolated microglia, P35 brain
Organism Mus musculus
Characteristics age: postnatal day 35
Sex: female
index: TGACCA
strain: C57BL6/J
group: Mother given maternal allergic asthma inflammation on gestation day 9,12, and 17
Treatment protocol Briefly, on postnatal day (P) 42 and 49 sexually naive female C57Bl/6J mice were sensitized with a single intraperitoneal injection of 10μg ovalbumin (OVA, Sigma, St. Louis, MO USA) in 1mg (Al)OH3 (InvivoGen, San Diego, CA USA) dissolved in 200μl phosphate buffered saline (PBS). One week following the second sensitization treatment, female mice were mated overnight and checked daily for the presence of a seminal plug, noted as gestational day 0.5 (G0.5). Pregnant mice were randomly assigned to either the allergic asthma or control group and exposed to either an aerosolized solution of 1% (wt/vl) OVA in PBS (maternal allergic asthma (MAA) group) or vehicle control for three 45-minute induction on gestational days 9.5, 12.5, and 17.5, to correspond with early, middle, and late gestation. Following the final induction, mice were returned to their home cages, single housed, and left undisturbed until the birth of their litters. Pups remained with their mother until weaning on P21, at which time the offspring were group housed with same-sex littermates.
Extracted molecule genomic DNA
Extraction protocol P35 female mice were deeply anaesthetized with CO2 and then quickly perfused intracardially with ice-cold PBS. Whole brains were removed and stored on ice in HBSS w/out Ca/Mg until processed. Each brain was gently homogenized to a single cell solution using a dounce, then added to 1.8ml of isotonic Percoll to form a 30% isotonic Percoll solution. Isotonic Percoll gradients were then constructed with layers of 70% with 1x phenol red, 37% and the top layer of brain/30%. The resulting gradients were spun in a swinging bucket centrifuge for 30min at 500xg at room temperature. The top layer of myelin and non-microglial cells were discarded and the microglia at the interface between the 37% and 70% layers were collected. The resulting microglia were then washed in HBSS and counted. An additional animal was run in parallel and harvested microglia were used to assess purity by flow cytometry for CD45.2 and CD11b. The collected cells were immediately flash frozen for RNA/DNA extraction.
DNA was isolated from the microglia samples using ZR-Duet RNA/DNA kit (Zymo, D7001). WGBS libraries were constructed using the Illumina EpiGnome/TruSeq DNA Methylation kit (Illumina, EGMK81312) with indexing barcodes (Illumina, EGIDX81312). 100 ng of DNA from each sample was bisulfite converted using the EZ DNA Methylation-Lightning kit (Zymo, D5030. Each library was given a unique barcode identifier and 14 cycles of PCR amplification. Libraries were quantified by Quibt and quality was assessed by Bioanalyzer.
Sequencing of barcoded and pooled libraries was performed across two lanes of two runs on the Illumina HiSeq4000 to obtain 100 bp single end reads.
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina HiSeq 4000
Description Mother given maternal allergic asthma inflammation on gestation day 9,12, and 17
Data processing Filter using Illumina flag
Separate and trim reads based on adapter contamination
Alignto mouse genome (mm10) using the Bisulfite Seeker2 program (v2.0.8 with bowtie1.1.1)
Combine aligned reads, both trimmed and no adapter contamination
Convert sam files to percent methylation bed files
Combine all chromosome percent methylation bed files into a single file
Convert bed file to bigwig
Genome_build: mm10
Supplementary_files_format_and_content: percent methylation in bigwig
Submission date Feb 06, 2017
Last update date May 15, 2019
Contact name Annie Vogel Ciernia
Phone 6048270752
Organization name University of British Columbia
Street address 2215 Wesbrook Mall room 4550, Centre for Brain Health
City Vancouver
State/province British Columbia
ZIP/Postal code V6T 2A1
Country Canada
Platform ID GPL21103
Series (2)
GSE94568 Microglia isolated from juvenile offspring of dams with allergic asthma exhibit methylation and transcriptional alterations to autism risk genes [WGBS]
GSE94569 Microglia isolated from juvenile offspring of dams with allergic asthma exhibit methylation and transcriptional alterations to autism risk genes
BioSample SAMN06308214
SRA SRX2540539

Supplementary file Size Download File type/resource 130.6 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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