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| Status |
Public on Nov 01, 2019 |
| Title |
Young adult OPCs rep 3 |
| Sample type |
SRA |
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| Source name |
A2B5 positive brain cells
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| Organism |
Rattus norvegicus |
| Characteristics |
cell type: Oligodendrocyte progenitor cells
age: 2-3 months
strain: Sprague Dawley
gender: female
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| Growth protocol |
All cells were isolated from female Sprague Dawley rats that were housed under standard laboratory conditions on a 12 h light/dark cycle with constant access to food and water. All animals were housed in pairs or groups of up to 4 animals.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Adult male and female rats (2-24 months) were decapitated after lethal injection with phenobarbital. The brains were removed quickly and placed into ice-cold isolation medium (SI Tab. 3, alternatively Hibernate A Brainbits). The telencephalon and cerebellum were dissected in isolation medium; meninges, and the olfactory bulb were mechanically removed and the brain tissue was mechanically minced into 1mm3 pieces. The tissue pieces were spun down at 100g for 1min at RT and the tissue was washed in HBSS- (no Mg2+ and Ca2+, Gibco). Each half of the brain was mixed with 5ml of dissociation solution (34U/ml papain (Worthington), 20μg/ml DNAse Type IV (Gibco) in isolation medium). The brain tissue was dissociated on a shaker (50rpm) for 40 min at 35ºC. The digestion was stopped by addition of ice cold HBSS-. The tissue was centrifuged (200g, 3 min, RT), the supernatant completely aspirated and the tissue resuspended in isolation medium supplemented with 2% B27 and 2mM sodium-pyruvate (trituration solution). The tissue was allowed to sit in this solution for 5min. To obtain a single cell suspension the tissue suspension was triturated 10 times using first a 5ml serological pipette and subsequently three fire polished glass pipettes (opening diameter >0.5mm). After each trituration step the tissue suspension was allowed to sediment (approximately 1-2 min) and the supernatant (approximately 2ml), containing the cells, was transferred into a fresh tube. After each round of trituration 2ml of fresh trituration solution were added. To remove accidentally transferred undigested tissue bits, the collected supernatant was filtered through 70μm cell strainers into tubes that contained 90% isotonic Percoll (GE Healthcare, 17-0891-01, in 10xPBS pH7.2 (Lifetech). The final volume was topped up with phenol-red free DMEM/F12 with HEPES (Gibco) and mixed to yield a homogenous suspension with a final Percoll concentration of 22.5%. The single cell suspension was separated from remaining debris particles by gradient density centrifugation (800g, 20min, RT, without break). The myelin debris and all layers without cells were discarded and the brain cell containing phase (last 2ml) and cell pellet were resuspended in HBSS+ and combined in a fresh 15ml tubes and centrifuged (300g, 5min, RT). The cell pellet was resuspended in red blood cell lysis buffer (Sigma, R7757) and incubated for 1min at RT to remove red blood cells. 10ml of HBSS+ were added to this cell suspension and spun down (300g, 5min, RT). The cell pellets were resuspended in 0.5ml modified Milteny washing buffer (MWB, 2mM EDTA, 2mM Na-Pyruvate, 0.5% BSA in PBS, pH 7.3) supplemented with 10ng/ml human recombinant insulin (Gibco). To this cell suspension 2.5µg mouse-anti-rat-A2B5-IgM antibody (Millipore, Extended data Tab. 4) were added for every 10 million cells. After 25 min incubation, gently shaking at 4ºC, 7ml of MWB were added. The solution was centrifuged (300g, 5min, RT) and the pellet resuspended in 80μl MWB supplemented with 20μl rat-anti-mouse-IgM antibody (Milteny, 130-047-302) per 10 million cells. The cells were incubated for 15 min, slowly shaking at 4ºC. The secondary antibody was again washed out with 7ml MWB and the sample was centrifuged (300g, 5min, RT). The cell pellet was resuspended in 0.5ml and MACS was performed according to the recommendations of the supplier. Briefly, a MS column (Milteny, 130-042-201) were inserted into MiniMACS Separator (Miltenyi; 130-042-102) and pre-wet with 0.5ml MWB. Resuspended cells were put onto one MS column. Subsequently the column was washed three times using 500μl MWB for each wash. Finally, A2B5 positive cells were flushed out the column with 1ml MWB. The cells were pelleted by centrifugation and the RNA was isolated using the Qiagen RNAeasy micro kit (Qiagen).
Purification of polyA+ mRNA
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
RNA depleted from ribosomal RNA
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| Data processing |
Reads were quality-trimmed using 'TrimGalore!'.
Trimmed reads were aligned to the rat reference genome (Rnor_6.0/rn6) using 'tophat2' with paramers "--no-coverage-search --read-mismatch 2 --max-multihits 1 --b2-sensitive", allowing for two mismatches and considering uniquely mapping reads only.
Read counts per ENSEMBL 92 transcripts were obtained using featureCounts' with options '-p -O -C --minOverlap 10'
Genome_build: RGSC Rnor_6.0/rn6
Supplementary_files_format_and_content: Read counts per ENSEMBL 92 obtained by 'featureCounts'.
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| Submission date |
Jul 24, 2019 |
| Last update date |
Nov 01, 2019 |
| Contact name |
Sabine Dietmann |
| Organization name |
Washington University School of Medicine
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| Street address |
4565 McKinley Avenue
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| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL22396 |
| Series (1) |
| GSE134765 |
Transcriptome analysis of freshly isolated young and aged rat oligodendrocyte progenitor cells (OPCs) |
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| Relations |
| BioSample |
SAMN12348980 |
| SRA |
SRX6578991 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3967162_OPC.young.3.counts.txt.gz |
2.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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