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Sample GSM7233997

Query DataSets for GSM7233997

Status

Public on Apr 30, 2024

Title

RNA_UN_1

Sample type

SRA

Source name

Mid thoracic spinal cord

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue: spinal cord
condition: uninjured
genotype: wild type

Extracted molecule

total RNA

Extraction protocol

Single-nucleus dissociation of the mouse lumbar spinal cord was performed according to our established procedures. Following euthanasia by isoflurane inhalation and cervical dislocation, the spinal cord injury site was immediately dissected and frozen on dry ice. Spinal cords were doused in 500 µL sucrose buffer (0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl2, 3 mM Mg acetate, 0.1 mM EDTA, 1 mM DTT) and 0.1% Triton X-100 with the Kontes Dounce Tissue Grinder. 2 mL of sucrose buffer was then added and filtered through a 40 µm cell strainer. The lysate was centrifuged at 3200 g for 10 min at 4°C. The supernatant was then decanted, and 3 mL of sucrose buffer was added to the pellet for 1 min. We homogenized the pellet using an Ultra-Turrax and 12.5 mL of density buffer (1 M sucrose, 10 mM HEPES [pH 8.0], 3 mM Mg acetate, 1 mM DTT) was added below the nuclei layer. The tube was centrifuged at 3200 g at 4°C and supernatant poured off. Nuclei on the bottom half of the tube wall were collected with 100 µL PBS with 0.04% BSA and 0.2 U/µL RNase inhibitor. Finally, nuclei were resuspended through a 30 µm strainer, and adjusted to 1000 nuclei/µL. snRNA-seq library preparation was carried out using the 10x Genomics Chromium Multiome ATAC + Gene Expression Kit Version 3. The nuclei suspension was added to the Chromium RT mix to achieve loading numbers of 10,000 nuclei. For downstream cDNA synthesis (13 PCR cycles), library preparation and sequencing, the manufacturer’s instructions were followed.
We used 10x Chromium Single Cell Multiome ATAC + Gene Expression kit to profile the mid thoracic spinal cord of mice.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

10x v3

Data processing

Reads were aligned using Cellranger arc v.1.0.1. For snRNA-seq, the final data matrix is generated as per Cellranger arc pipeline. For snATAC-seq, both gene-level and peak matrices are generated following Snapatac pipeline. The gene-level features are generated based on GENCODE vM10 and the peak features are derived using MACS2.
Data were analyzed using Seurat v3
Assembly: mm10-1.2.0 (GRCm38.101)
Supplementary files format and content: tab-delimited text files include UMI values for each cell with tab delimeters

Submission date

Apr 27, 2023

Last update date

Apr 30, 2024

Contact name

Alan Yue Yang Teo

E-mail(s)

yueyang.teo@epfl.ch

Organization name

EPFL

Department

Lab