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| Status |
Public on Jun 30, 2019 |
| Title |
MCAO2_CD163 |
| Sample type |
SRA |
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| Source name |
sorted cells from brain after MCAO
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Wistar
developmental stage: adult
tissue: brain
treatment: Rats were induced 90 min of transient MCAO
cell markers: CD11b+CD163+
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| Treatment protocol |
Focal cerebral ischemia was induced by transient occlusion of middle cerebral artery (MCA) as previously reported (Szydlowska, Zawadzka, & Kaminska, 2006; Zawadzka et al., 2012). Briefly, the rats were anesthetized using 1.5% isoflurane in oxygen and ischemia was produced by advancing the tip of a rounded 0.4 mm nylon suture (Doccol Co.) into the right internal carotid artery entering through the right external carotid artery. After 90 min of occlusion, the nylon filament was withdrawn to allow reperfusion. Throughout the surgery the animal’s core body temperature was maintained at 37±0.5°C with a thermostatically controlled heating pad. For the Sham operated animals the right common carotid artery was isolated and the external carotid artery was permanently ligated.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
We used CD163 as a marker of perivascular and meningeal macrophages in rats, whereas in mice CD206 and CD169 identify perivascular and meningeal macrophage population in the CNS. In rats we found an accumulation of CD163+ perivascular and meningeal macrophages in response to cerebral ischemia, which tend to infiltrate in to the parenchymal lesion. RNA-sequencing of FACS sorted CD11b+CD163+ population revealed these cells are highly proliferative and express pro-inflammatory phenotype in the parenchyma
In total, 6 strand-specific RNA libraries for high-throughput sequencing were prepared (three biological replicates for each treatment) using the KAPA Stranded mRNA Sample Preparation Kit according to the manufacturer’s protocol. Briefly, the poly-A containing mRNA molecules were purified from 500ng of total RNA (extracted FACS sorted cells from the brain samples of sham operated and MCAo animals) using poly-T oligo-attached magnetic beads (Kapa Biosystems, MA, USA ) The mRNA was then fragmented and the first-strand cDNA was synthesized using reverse transcriptase and random hexamers. Second cDNA synthesis was performed by removing the RNA template and synthesizing a replacement strand, incorporating dUTP in place of dTTP, to generate double-stranded (ds) cDNA. dsDNA was then subjected to the addition of “A” bases to the 3′ ends and ligation of adapters from NEB followed by uracil digestion in a loop structure of adapter by USER enzyme from NEB (Ipswich, MA, USA). Amplification of fragments with adapters ligated on both ends was performed by PCR using primers containing Truseq barcodes from NEB (Ipswich MA, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Basecalling with HCS 2.2.68
Bcl2fastq.gz processing of bcl files to fastq.gz
fastq.gz trimming for adapter contamination and low quality reads and aligned with tophat2 to rn5.fa reference
FPKM calculation with RSEM software using Ensembl transcriptome annotations for rn5.fa genome version
Genome_build: rn5
Supplementary_files_format_and_content: normalized abundance measurements
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| Submission date |
May 23, 2017 |
| Last update date |
Jun 30, 2019 |
| Contact name |
Bartosz Wojtas |
| E-mail(s) |
bartoszwojtas83@gmail.com
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| Phone |
692156006
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| Organization name |
Nencki Institute of Experimental Biology
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| Street address |
Pasteura 3
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| City |
Warsaw |
| ZIP/Postal code |
02-793 |
| Country |
Poland |
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| Platform ID |
GPL18404 |
| Series (1) |
| GSE99206 |
Perivascular macrophages proliferate in the CNS, but also contributed by bone-marrow precursors in response to cerebral ischemia |
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| Relations |
| BioSample |
SAMN07157039 |
| SRA |
SRX2844874 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2635757_MCAO2_CD163_RSEM.txt.gz |
360.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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