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| Status |
Public on Dec 20, 2023 |
| Title |
Late, Ctrl, replicate1, scRNAseq |
| Sample type |
SRA |
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| Source name |
Spinal Cord
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| Organism |
Mus musculus |
| Characteristics |
timepoint: Late
sequencing id: P25316_1002
tissue: Spinal Cord
cell line: Sox10:Cre-RCE:LoxP (EGFP)
replicate: 1
genotype: mm10
debris removal: percoll
batch: 5
Sex: pooled male and female
data tissue_collection: 3/9/2022
number of_mice: 2
treatment: CFA-Ctrl
eae scores: 0/0
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| Treatment protocol |
For the EAE induction, animals were injected subcutaneously with an emulsion of MOG35–55 in complete Freund's adjuvant (CFA) (Hooke Laboratories, EK-2110, containing 1 mg MOG35-55/mL emulsion and 2-5 mg killed mycobacterium tuberculosis H37Ra/mL emulsion, Hooke Laboratories adjusted concentrations by lot) followed by the intraperitoneal injection of pertussis toxin in PBS on day 0 and day 1 (200-225 ng per animal, adjusted by lot according to Hooke manufacturer’s instructions). Scores of EAE were graded according to the following criteria: 0, asymptomatic; 1, limp tail or titubation; 2, limp tail and weakness of hindlimbs; 3, limp tail and complete paralysis of hindlimbs; 4, limp tail, complete paralysis of two hindlimbs with forelimb involvement; 5, moribund or dead; 0.5 for intermediate symptoms. CFA-control mice were injected subcutaneously with the control emulsion containing CFA but without MOG35–55 (Hooke Laboratories, CK-2110), and followed by the administration of pertussis toxin in PBS on day 0 and day 1 (200-225 ng per animal, adjusted by lot according to Hooke manufacturer’s instructions). Spinal cords from EAE and CFA-Ctrl were collected at early (day 8-9), peak (day 14-15), and late stage (day 38-40)
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| Extracted molecule |
total RNA |
| Extraction protocol |
At early, peak, and late stage, mice were perfused with PBS, and spinal cords were collected. Spinal cord tissues were then dissociated into a single cell suspension according to the manufacturer’s protocol of Adult Brain Dissociation Kit, mouse and rat (Miltenyi Biotec, 130-107-677, we did not perform the red blood cells removal step since majority of the red blood cells had been removed with PBS perfusion). For the debris removal step, majority of the samples were processed with 38% percoll except 2 EAE peak stage samples, and 2 EAE late stage samples were processed debris removal solution (Miltenyi Biotec, 130-107-677) according to the manufacturer’s protocol to enrich more OPCs. Spinal cord single EGFP+ cells were enriched with the BD FACSAria III Cell Sorter (BD Biosciences). The cells were then lysed and washed according to the demonstrated protocol, Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing (10x Genomics, CG000365), with modifications: the cells were centrifuged for 10 min at 300 g and 4°C, resuspended in lysis buffer (containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.01% IGEPAL (CA-630), 0.001% Digitonin, 1% BSA, 1 mM DTT, 1 U/ul RNase inhibitor) and incubated on ice for 3 min. After the incubation, wash buffer (containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT, 1 U/ul RNase inhibitor) was added on top without mixing. The nuclei were centrifuged for 5 min at 500 g and 4°C. Nuclei were washed once in wash buffer followed by another wash with diluted 1x Nuclei buffer (10x Genomics, PN-1000283) containing 1% BSA, 1 mM DTT and 1 U/ul RNase inhibitor.
Libraries were sequenced on an Illumina Novaseq 6000 with a 50-8-24-49 read setup for ATAC (minimum 25,000 read pairs per cell) and a 28-10-10-90 read setup for RNA (minimum 20,000 read pairs per cell).
The Chromium Next GEM Single Cell Multiome ATAC + Gene Expression chemistry (10x Genomics, PN-1000283) was applied to create single cells ATAC and RNA libraries. 26 EAE mice (8 mice from early stage, 8 mice from peak stage and 10 mice from late stage), 12 CFA-Ctrl mice (4 mice from early stage, 4 mice from peak stage and 4 mice from late stage) were used for independent replicates.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10X Genomics
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| Data processing |
A total of 6 batches were collected from the sequencing facility. Fastq files from 19 samples were processed throughout the 10X genomics standard pipeline. Gene expression and chromatin accessibility libraries were input into cellranger-arc ‘count’ v2.0.2 with default settings to align the biological readouts on the associated mm10 reference genome v2020-A-2.0.0. Sample aggregation of both transcriptomic and genomics metrics was done using the ‘aggr’ of the same cellranger executable file, without normalization. The aggregated count matrix and fragments file were loaded into R v4.2.3. The former was lodged into a Seurat v4.3.056 assay while the latter accommodate into a Signac v1.9.057 chromatin assay associated to an Ensembl58 base annotation v79 for mouse where UCSC nomenclature was applied to provide gene names readability. If not specifically mentioned, the following depictions of the analysis pipeline belong to either Seurat or Signac packages. After assessing that each cell could be uniquely identified, quality metrics for both gene expression and peak accessibility were calculated. Among others, the mitochondrial ratio, the cell cycle score, the nucleosome signal, and the Transcription Start Site (TSS) enrichment were generated to support the following quality control cutoffs. Depending of the quality of each sample, for each cell, a maximum of 30,000/150,000 and a minimum of 1,000 ATAC counts, a maximum of 10,000/45,000 and a minimum of 600 RNA counts, a minimum of 600 detected genes, a maximum of 1/1.5 nucleosomal signal, a TSS minimum enrichment of 2 and a maximum percentage of mitochondrial information of 15/25 were prerequisite to consider a given cell for the downstream analysis. These thresholds shrinked by around 12% the overall number of cells, from 104,479 down to 91,757. While these thresholds are on purpose not too strained, they did not lead to any clustering perturbation downstream.
Assembly: mm10
Supplementary files format and content: EAE_multiomics.rds : Aggregation of cells from all samples in a Seurat object with associated metadata
Supplementary files format and content: EAE_multiomics_fragments.tsv.gz : Aggregation of fragments from all samples
Supplementary files format and content: EAE_multiomics.bedpe : Peaks to genes links using LinkPeaks function from Signac package with 50,000bp maximum distance from TSS and minimum 10 cells to validate the link
Supplementary files format and content: Astro_Ctrl-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of Astrocytes population at Ctrl time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: Astro_Early-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of Astrocytes population at Early time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: Astro_Peak-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of Astrocytes population at Peak time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: Astro_Late-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of Astrocytes population at Late time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: OPC_Ctrl-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of OPC population at Ctrl time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: OPC_Early-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of OPC population at Early time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: OPC_Peak-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of OPC population at Peak time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: OPC_Late-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of OPC population at Late time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: COP_Ctrl-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of COP population at Ctrl time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: COP_Peak-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of COP population at Peak time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: COP_Late-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of COP population at Late time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: Micro_Ctrl-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of Microglia population at Ctrl time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: Micro_Early-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of Microglia population at Early time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: Micro_Peak-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of Microglia population at Peak time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: Micro_Late-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of Microglia population at Late time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL1_Ctrl-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL1 population at Ctrl time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL1_Early-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL1 population at Early time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL1_Peak-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL1 population at Peak time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL1_Late-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL1 population at Late time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL2_Ctrl-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL2 population at Ctrl time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL2_Early-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL2 population at Early time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL2_Peak-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL2 population at Peak time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL2_Late-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL2 population at Late time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL56_Ctrl-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL56 population at Ctrl time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL56_Early-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL56 population at Early time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL56_Peak-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL56 population at Peak time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
Supplementary files format and content: MOL56_Late-TileSize-100-normMethod-rc.bw : Bigwig file of fragments coverage in 100bp window size of MOL56 population at Late time point normalized by relative count (rc) as a scaling factor of 10,000 divided by the total number of fragments involved in this file
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| Submission date |
Dec 19, 2023 |
| Last update date |
Dec 20, 2023 |
| Contact name |
Bastien Hervé |
| E-mail(s) |
bastienherve61@gmail.com
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| Organization name |
Karolinska Institutet
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| Department |
MBB
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| Lab |
Castelo-Branco
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| Street address |
Solnavägen 9
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| City |
Solna |
| ZIP/Postal code |
17165 |
| Country |
Sweden |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE250589 |
Distinct transcriptomic and epigenomic responses of mature oligodendrocytes during disease progression in a mouse model of multiple sclerosis |
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| Relations |
| BioSample |
SAMN38983875 |
| SRA |
SRX22966178 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7982342_P25316_1002_filtered_feature_bc_matrix.h5 |
137.3 Mb |
(ftp)(http) |
H5 |
| GSM7982342_P25316_1002_gex_molecule_info.h5 |
251.4 Mb |
(ftp)(http) |
H5 |
| GSM7982342_P25316_1002_raw_feature_bc_matrix.h5 |
169.9 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
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