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Sample GSM4665462 Query DataSets for GSM4665462
Status Public on May 21, 2021
Title Ctr1
Sample type SRA
 
Source name spinal cord tissue
Organism Mus musculus
Characteristics tissue: spinal cord tissue
disease state: control
Treatment protocol For the induction of EAE, the mouse model of MS, animals were injected subcutaneously with an emulsion of MOG35-55 peptide in complete Freud’s adjuvant (CFA; EK-2110 kit from Hooke Laboratories) followed by intraperitoneal injection with pertussis toxin in PBS (200 ng per animal) on the day of immunization and with 24 hours delay (according to manufacturer’s instructions). Control animals underwent the same treatment, but CFA without MOG35-55 peptide (CK-2110 kit from Hooke Laboratories) was used instead. Spinal cord and brains were collected at the peak of the disease when clinical score 3 has been reached, which corresponds to limp tail and complete paralysis of hind legs. Animals that did not reach this clinical score were not analyzed in this study.
Extracted molecule genomic DNA
Extraction protocol Mice were sacrificed with a ketaminol/xylazine intraperitoneal injection followed by intracardiac perfusion with PBS where after brain and spinal cords were collected and dissociated using the adult brain dissociation kit (130-107-677, Miltenyi) following manufacturer’s instructions including myelin debris removal, but excluding the red blood cell removal step.
Immediately after dissociation, cells were stained with DAPI and sorted on a FACS Aria III cell sorter (BD Biosciences). Sox10GFP+/DAPI- cells were collected in PBS + 0.5% BSA and pooled with Sox10GFP-/DAPI- cells with a 4:1 ratio. The pool of cells was then lysed and washed according to the Demonstrated Protocol: Nuclei Isolation for Single cell ATAC Sequencing (10x Genomics) as follows: the cells were centrifuged for 10 minutes at 300xg and 4°C, resuspended in ATAC lysis buffer (containing 0.1% IGEPAL (CA-630), 0.1% Tween-20, 0.01% Digitonin, 1% BSA, 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) and incubated on ice for 3 minutes. After the incubation, wash buffer (containing 0.1% Tween-20, 1% BSA, 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) was added on top without mixing and the nuclei were centrifuged for 5 min at 500xg and 4°C. Nuclei were washed once in Diluted Nuclei buffer (10x Genomics) containing 1% BSA, before adding to the tagmentation mix (10x Genomics). The Chromium Single Cell ATAC v1 Chemistry was used to create single-cell ATAC libraries. Two EAE and four CFA-control animals were used for independent replicates. Libraries were sequenced on an Illumina Novaseq 6000 with a 50-8-16-49 read set-up and a minimum of 25 000 read pairs per cell. 
Chromium Single Cell ATAC v1 Chemistry
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
Description unbiased total nuclei isolation
Data processing Chromiun Single Cell ATAC v1 Chemistry was used to create the scATAC-seq libraries. Samples were sequenced in Illumina NovaSeq and demultiplexed. Demultiplexed samples were converted to fastq files and aligned to mm10 reference assembly and process using Cellranger-ATAC count (version 1.2.0) and cellranger-atac aggr. Normalizing the sequencing depth.
Cellranger-atac aggr output bam files, peaks file and fragment file were used as input for Seurat/Signac v0.25 R packages for downstream analysis.
Final cells were selected following the QC parameters below, min peak region 1000 fragments and maximun 20000 resulting in 4895 cells.
Cells were clustered based on peaks and annotated using reference annotations from Falcao, van Bruggen et al. 2018 Nature Medicine.
Genome_build: mm10
Supplementary_files_format_and_content: The bam files belong to individual samples as bam files from Cellranger ATAC v1.2.0 preprocessing.
Supplementary_files_format_and_content: fragments.tsv, fragments coordenates including cell barcode ID and number of duplicate reads pairs per fragment. peaks.bed refers to scATAC peaks that were called individually for each sample and then merged, samples were normalized to depth with cellranger ATAC v1.2.0 aggr.
Supplementary_files_format_and_content: Meijer_Agirre_scATAC_metadata_umap.csv.gz file includes scATAC-seq metadata, including QC metrics, cluster/celltype annotations and UMAP coordenates in csv file format. Where columns are cell Ids and rows peak coordenates.
Supplementary_files_format_and_content: Meijer_Agirre_EAEscATAC_signac025.Robj belongs to the R object from Seurat/Signac.0.25 R packages including gene activity matrices and peak matrices used in the study.
 
Submission date Jul 10, 2020
Last update date Jan 04, 2022
Contact name Eneritz Agirre
Organization name Karolinska Institutet
Department MBB
Lab Castelo-Branco, Molecular Neurobiology
Street address Solnavägen 9
City Stockholm
ZIP/Postal code 17165
Country Sweden
 
Platform ID GPL24247
Series (2)
GSE154175 A primed immune transcriptional program is activated in oligodendroglia in multiple sclerosis [SCATAC_10X]
GSE166179 A primed immune transcriptional program is activated in oligodendroglia in multiple sclerosis
Relations
BioSample SAMN15502378
SRA SRX8706628

Supplementary file Size Download File type/resource
GSM4665462_P13503_1006_filtered_peak_bc_matrix.h5 33.8 Mb (ftp)(http) H5
GSM4665462_P13503_1006_raw_peak_bc_matrix.h5 43.5 Mb (ftp)(http) H5
GSM4665462_singlecell_P13503_1006.csv.gz 4.6 Mb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record
Processed data provided as supplementary file

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