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| Status |
Public on Sep 12, 2024 |
| Title |
GDMPON_2, Myrf fl/fl PLP CreERT WT/WT, snRNAseq |
| Sample type |
SRA |
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| Source name |
Optic Nerve
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| Organism |
Mus musculus |
| Characteristics |
tissue: Optic Nerve
strain: C57/Bl6
Sex: Female
genotype: WT
age (weeks): 18
agent: tamoxifen
time: 10 weeks
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Blood was removed from deeply anesthetized mice (360mg/kg ketamine, 48mg/kg xylazine) via intracardiac perfusion with 10mL 1x PBS. Optic nerves were dissected out and immediately snap-frozen on dry ice. Frozen tissue was stored at -80°C for up to six months, until subsequent tissue processing. The nuclei isolation buffer (NIB, includes 146 mM NaCl, 5 mM Tris-HCl, 1 mM CaCl2, 21 mM MgCl2, 0.03% Tween-20, 0.01% BSA, 1 µg/mL actinomycin D, pH 7.5) was prepared with 1 tablet of protein inhibitor cocktail (cOMPLETE Mini lacking EDTA, Sigma) along with 15uL of RNAsin (40U/µL) per 10 mL NIB. When optic nerves were removed from the freezer, they were immediately placed in 2mL NIB solution in a 7mL Dounce grinder and ground 20 times with a loose pestle (A). Then, the homogenate was passed through a 200 µm strainer. The homogenate was ground 10 times with a tight pestle (B), then 2 mL NIB was added and the homogenate was passed through a 40 µm strainer. The homogenate was ground 5 times with a tight pestle (B), then passed through a 20 µm filter. The sample was then centrifuged at 500 x g for 5 minutes at 4°C, the supernatant discarded, and the sample placed on ice. The pellet was resuspended in 1.5 mL of NIB, then centrifuged at 500 x g for 5 min at 4°C, the supernatant discarded, and the pellet resuspended in 0.5 mL Resuspension Buffer (NIB + 50uL SuperaseIN [20U / µL] + 1% BSA). The nuclei suspension was mixed 1:200 with RedDot (2.5uL). The samples were then isolated from debris by fluorescence-activated nuclei sorting (FANS). Two main gates were used: a 561+683 emission for the RedDot stain and a low trigger pulse width as singlet discriminator. 100,000 nuclei were aimed to be sorted. Following sorting, samples were centrifuged at 300 x g for 1 min at 4°C, held on ice for 1 minute then spun for 1 minute at 300 x g at 4°C. The top supernatant was carefully removed and loaded onto the 10x microfluidics chromium chips.
snRNAseq was performed using a Chromium Next GEN Single Cell 3′ Reagent Kit v3.1 (10x Genomics). Single nuclei were partitioned in droplets with single Gel Beads, which contained primers with cell-tagging indexes. Single nucleus suspensions with concentration of at least ~3000/µl were loaded targeting 10,000 nuclei per sample. The resulting cDNA was profiled on a Bioanalyzer NanoChip (Agilent), then used as a template for library preparation according to the reagent kit protocol. (https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/library-prep/chromium-single-cell-3-reagent-kits-user-guide-v-3-1-chemistry).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
Fastq files were prepared using bcl2fastq (Illumina) and then aligned to a reference genome using Cellranger v. 7.0.0 (10x Genomics). Reads were mapped to both exonic and intronic regions to include the pre-mRNA transcriptome.
Ambient RNA contamination was estimated and removed using SoupX v. 1.6.2
Mitochondrial features were manually removed by excluding all features starting with mt-
The normalized count matrix integration, batch effect correcting, and cell clustering were performed using Seurat v. 4.3.0
Nuclei were filtered based on 1,000 < nFeature_RNA < 4,000 as well as 1,250 < nCount_RNA < 10,000
Assembly: mm10
Supplementary files format and content: Raw feature-barcode matrices from each replicate
Supplementary files format and content: Filtered feature-barcode matrices from each replicate
Library strategy: snRNA-seq
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| Submission date |
Sep 22, 2023 |
| Last update date |
Sep 12, 2024 |
| Contact name |
Ben Emery |
| Organization name |
Oregon Health & Science University
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| Department |
Neurology
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| Street address |
3181 SW Sam Jackson Park Road
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| City |
Portland |
| State/province |
Oregon |
| ZIP/Postal code |
97239 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE243788 |
snRNA-Seq of optic nerve from two models of inducible demyelination. |
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| Relations |
| BioSample |
SAMN37505763 |
| SRA |
SRX21855974 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7795663_GDMPON_2_filtered_feature_bc_matrix.h5 |
25.2 Mb |
(ftp)(http) |
H5 |
| GSM7795663_GDMPON_2_raw_feature_bc_matrix.h5 |
84.5 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
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