|
| Status |
Public on Jan 10, 2024 |
| Title |
Chronic EAE infiltrating, biol rep 3 |
| Sample type |
SRA |
| |
|
| Source name |
Spinal cord
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Spinal cord
disease state: Chronic EAE
cell type: CNS infiltrating myeloid cells
genotype: Cx3cr1YFPCreERT2:R26tdTomato
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mice were perfused with aCSF, spinal cords were extracted and underwent mechanical dissociation in homogenization buffer. After myelin removal, live cells were isolated via FACS based on the RFP or YFP protein expression.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, the cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10x Genomics
|
| Data processing |
The barcoded processing and gene counting were made using the Cell Ranger software v7.1.0 [Zheng2017] and post-alignment processing and visualisation was performed using Seurat v4.3.0.1 [Stuart2019]. Data was normalised using Sctransform [Hafemeister2019].
The clustering was performed using SLM; ClustAssess was used for identifying stable clusters; the identification of markers was performed per cluster vs its complement.
The visualisation and community accessible overview were complied using ShinyCell [Ouyang2021]
Assembly: GRCm38 (Ensembl 93 from pre-built Cell Ranger reference 3.1.0)
Supplementary files format and content: count matrix containing raw gene expression (csv)
|
| |
|
| Submission date |
Nov 17, 2023 |
| Last update date |
Feb 13, 2024 |
| Contact name |
Irina Mohorianu |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Cambridge
|
| Department |
Wellcome-MRC Cambridge Stem Cell Institute
|
| Street address |
Puddicombe Way
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0AW |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE248172 |
Mitochondrial complex I activity in microglia sustains neuroinflammation [scRNA-seq] |
| GSE248175 |
Mitochondrial complex I activity in microglia sustains neuroinflammation |
|
| Relations |
| BioSample |
SAMN38302087 |
| SRA |
SRX22560620 |
