|
Status |
Public on Dec 14, 2023 |
Title |
M-KO-MONO-Macrophages |
Sample type |
SRA |
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|
Source name |
Brain
|
Organism |
Mus musculus |
Characteristics |
tissue: Brain cell line: primary cells cell type: Macrophages genotype: CD163KO treatment: α-syn MONO
|
Treatment protocol |
For whole population RNA sequencing, mice (batch 1 and batch 2) received bilateral intrastriatal injection of α-syn MONO (Males WT n=7 & CD163KO n=4*; Females WT n=7 &CD163KO n=5), and α-syn PFF (Males WT n=7, & CD163KO n =5; Females WT n=7 & CD163KO n=5). Triplicates were used for sequencing. Mice were sacrificed 2 months post-surgery, their brains dissected and the immune cells (microglia & macrophages) isolated and FACS sorted for RNA purification and subsequent SMART-seq2 sequencing. *CD163KO-MONO males were excluded from the analysis due to inconsistencies in the technical replicates.
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Growth protocol |
Adult male and female CD163KO (CD163tm1.1(KOMP)Vlcg) mice and WT (C57BL/6N) littermates (3-4 months old) (n=179 total (n=95 males (50 WT + 45 CD163KO); n=84 females (43 WT+ 41 CD163KO)) were used in this study 1. Mice weighted 20-25g (12 weeks old) at the time of the surgery and were housed maximum 4-6 per cage, with ad libitum access to food and water, in a climate-controlled facility under 12h/12h night/daylight cycle.
|
Extracted molecule |
total RNA |
Extraction protocol |
Once sorted, microglia (200000-500000 cells) and macrophage (10000-30000 cells) cell suspensions (>90% purity after sorting) were centrifuged at 4°C and 400xg for 5 minutes, resuspended in RLT Plus Lysis buffer (QIAGEN, Germany) with 1% β-Mercaptoethanol and vortexed for 1 minute for cell lysis. The solution was further homogenized using a 1mL syringe and a 21gag needle. Total RNA was extracted using RNeasy® Mini Kit (QIAGEN) according to the manufacturer’s protocol. Total purified RNA was sent to BGI Hong Kong where Switching Mechanism at 5’ End of RNA Template (SMART-seq2) sequencing service was requested. Samples were tested for quality control using Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit). RNA integrity (RNI) >7 was considered suitable for sequencing. Gene expression analysis was performed through full population RNA sequencing using the SMART-seq2 method according to BGI’s standard methodology.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
DNBSEQ-G400 |
|
|
Data processing |
Reads mapped to rRNA were removed and raw data was obtained. The sequencing reads which contained low-quality, adaptor-polluted and high content of unknown base (N) reads processed were removed before downstream analyses. Clean reads were mapped to reference using Bowtie2, and then gene expression level was calculated for each sample with RSEM DESeq2 package was used to detect the DEGs, determining at a cutoff of FDR corrected p-value≤0.05 and |log2(fold change)|≥1 as DEGs. Principal component analysis (PCA) was performed on the Macrophage and Microglia population respectively based on the rlog of dds result from DESeq2 Assembly: mm10 Supplementary files format and content: tab-delimited text files contain counts and FPKM for each sample
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|
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Submission date |
Sep 19, 2023 |
Last update date |
Dec 14, 2023 |
Contact name |
Yonglun Luo |
E-mail(s) |
alun@biomed.au.dk
|
Phone |
0045-22411944
|
Organization name |
Aarhus University
|
Street address |
Wilhelm Meyers Allé 4
|
City |
aarhus |
ZIP/Postal code |
8000 |
Country |
Denmark |
|
|
Platform ID |
GPL28457 |
Series (1) |
GSE243536 |
Sex-dimorphic neuroprotective effect of CD163 in an alpha-synuclein mouse model of Parkinson’s disease |
|
Relations |
BioSample |
SAMN37458610 |
SRA |
SRX21827018 |