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| Status |
Public on Dec 20, 2017 |
| Title |
PBS2 RNAseq |
| Sample type |
SRA |
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| Source name |
acutely isolated microglia, P35 brain
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| Organism |
Mus musculus |
| Characteristics |
age: postnatal day 35
Sex: female
strain: C57BL6/J
index: GCCAAT
group: Mother given PBS on gestation day 9,12, and 17
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| 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.
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| Extracted molecule |
total RNA |
| 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.
Duet RNA/DNA kit (Zymo, D7001) with on-column DNase digestion (Qiagen, 79254) for RNA extraction. RNA and DNA were quantified using Qubit High Sensitivity assays. RNA quality was assessed by Bioanalyzer and only samples with RIN scores greater than 7 were used for library preparation. 60-100 ng of input RNA per sample were used for RiboGone rRNA removal (Clonetech, 634847). 2 ng of rRNA depleted RNA per sample was then used for library construction using the SMARTer Stranded RNA-Seq kit (Clontech, 634836) and 14 cycles of PCR amplification. Library quality was assessed using a bioanalyzer and quantified by Qubit high sensitivity assay.
All samples were barcoded, pooled, and run on a single sequencing lane of a HiSeq3000 to generate 50 bp single end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
Mother given PBS on gestation day 9,12, and 17
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| Data processing |
RNA-seq reads were analyzed for both quality and adapter contamination using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
All reads were trimmed using Trimmomatic using Illuminaclip single end adapter trimming, a removal of the first three bp from the 5’ as recommended for SMARTer libraries, and a 4 bp sliding window trim for quality scores less than 15. All remaining reads greater than 15 bp were then reanalyzed using FASTQC to verify quality.
RNA-seq reads were aligned to mouse genome mm10 using the splicing-aware aligner Tophat2 (v2.0.14/bowtie2.2.5, stranded, Single End) and reads aligning to each gene will were counted using FeatureCounts with exclusion of multimapping reads
Genome_build: mm10
Supplementary_files_format_and_content: FeatureCounts raw counts per gene
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| Submission date |
Feb 06, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Annie Vogel Ciernia |
| E-mail(s) |
annie.ciernia@ubc.ca
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| Phone |
6048270752
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| Organization name |
University of British Columbia
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| Street address |
2215 Wesbrook Mall room 4550, Centre for Brain Health
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| City |
Vancouver |
| State/province |
British Columbia |
| ZIP/Postal code |
V6T 2A1 |
| Country |
Canada |
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| Platform ID |
GPL21493 |
| Series (2) |
| GSE94525 |
Microglia isolated from juvenile offspring of dams with allergic asthma exhibit methylation and transcriptional alterations to autism risk genes [RNA-Seq] |
| GSE94569 |
Microglia isolated from juvenile offspring of dams with allergic asthma exhibit methylation and transcriptional alterations to autism risk genes |
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| Relations |
| BioSample |
SAMN06298776 |
| SRA |
SRX2538739 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2477586_PBS2_counts.txt.gz |
190.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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