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| Status |
Public on Jul 01, 2022 |
| Title |
MG_FACS_CON_PLX_SAL_Rep3 |
| Sample type |
SRA |
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| Source name |
brain striatum
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| Organism |
Mus musculus |
| Characteristics |
tissue: brain striatum
cell type: microglia
group: CON:PLX
treatment: SAL
Sex: male
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| Treatment protocol |
Lipopolysaccharide (LPS) was delivered by intraperitoneal injection to mice at a dose of 4500 endotoxin units / gram body weight. Microglia were extracted 18 hours after LPS or saline treatment.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Male mice (8-10 wk) were deeply anesthetized with ether, perfused with PBS, and brains extracted. The meninges were removed and then the frontal cortex and striatum were collected into 2 mL tubes with HBSS on ice. The anterior striatum was collected from a coronal slice anterior to 1.7 mm bregma by collecting the tissue surrounding the nucleus accumbens. The next coronal slice was cut at -0.5 bregma and the dorsal and ventral striatum was excised from this slice, excluding the olfactory tubercle, and all striatum pieces were pooled together. The brain pieces were diced with scissors and then homogenized with papain according to the adult neural dissociation kit (Miltenyi). Briefly, the tissue was treated with papain and DNase at 37°C for 30 min with rotation. The homogenates were pipetted gently and filtered with a 70 µm strainer. The cell pellet was resuspended in debris removal solution (300 µL debris removal, 700 µL PBS), overlaid with 300 µL of PBS, centrifuged at 3000xg for 10 min, debris aspirated, washed with PBS, and centrifuged at 1000xg for 10 min. The cells were resuspended in 0.5% BSA in HBSS. For fluorescence-activated cell sorting the cells were stained with CD45-APC, CD11b-BV421, and P2RY12-PE antibodies for 30 min at 4C. The cells were washed with buffer and sorted for live (PI-), CD11b+,CD45-low, P2RY12+ before final collection into RNA lysis buffer. RNA was collected according to the kit instructions (Qiagen, Micro-RNeasy, PicoPure RNA isolation Kits).
RNA samples were converted to double stranded cDNA using the Ovation RNA-Seq System v2.0 kit (Tecan, Männedorf, Switzerland), which utilizes a proprietary strand displacement technology for linear amplification of mRNA without rRNA/tRNA depletion as per the manufacturer’s recommendations. This approach does not retain strand specific information. Quality and quantity of the resulting cDNA were monitored using the Bioanalyzer High Sensitivity kit (Agilent) which yielded a characteristic smear of cDNA molecules ranging in size from 500 to 2000 nucleotides in length. After shearing 500 nanograms of cDNA to an average size of 250 nucleotides with the Covaris S4 (Covaris Inc., Woburn, MA) library construction was completed with the TruSeq Nano kit (Illumina; San Diego, CA) according to the manufacturer’s instructions. mRNA libraries were sequenced on an Illumina Novaseq 6000 instrument using 150bp paired-end dual indexed reads and 1% of PhiX control.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
CPS3
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| Data processing |
Alignment: FASTQ files aligned and assembled with RSEM and STAR: rsem-calculate-expression -p 4 --paired-end --star --star-output-genome-bam --calc-ci --forward-prob 0.5
Counts: Count matrix was assembled using tximport in R files <- list.files(path = "../1.align/genes.results/") names(files) <- strtrim(files, 4) txi.rsem <- tximport(files, type = "rsem", txIn = FALSE, txOut = FALSE) txi.rsem$length[txi.rsem$length == 0] <- 1 table(txi.rsem$length == 0) save(txi.rsem, file = "txi.rsem.RData")
Differential expression was performed with DESeq2.
Assembly: GRCm38vM25
Supplementary files format and content: Adult_Rescue_counts.csv: Comma-separated file of raw counts from RSEM output.
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| Submission date |
Apr 28, 2022 |
| Last update date |
Jul 01, 2022 |
| Contact name |
Lindsay N Hayes |
| E-mail(s) |
lhayes14@jhmi.edu
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| Organization name |
Johns Hopkins University
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| Department |
Neuroscience
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| Street address |
600 North Wolfe Street, Meyer 4-136
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| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21287 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE201816 |
Prenatal immune stress blunts microglia reactivity which impairs neurocircuitry [Adult_Rescue] |
| GSE201817 |
Prenatal immune stress blunts microglia reactivity which impairs neurocircuitry |
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| Relations |
| BioSample |
SAMN27959501 |
| SRA |
SRX15041498 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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