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Sample GSM8268585

Query DataSets for GSM8268585

Status

Public on May 22, 2024

Title

left_hemisphere,WT,old_mouse_2

Sample type

SRA

Source name

left_hemisphere

Organism

Mus musculus

Characteristics

tissue: left_hemisphere
genotype: WT
exit age: 11m 26d
Sex: M
mouse: old_mouse_2

Extracted molecule

total RNA

Extraction protocol

Mice were culled via cervical dislocation and brains were dissected, with the olfactory bulbs and cerebellum being removed.The desired area of the brain were collected in ice-cold HBSS (w/o Ca2+ and Mg2+; 14175-053; Gibco) with 5% trehalose (T0167; Sigma Aldrich) and 30 µM actinomycin D (A1410; Sigma Aldrich) and were finely minced using a 22A scalpel. Brains were digested using the Adult Brain Dissociation Kit (130-107-677; Miltenyi Biotec), with the following modifications: (i) tissues were dissociated as described in the “manual dissociation” section of the Neural Tissue Dissociation Kit protocol (130-092-628; Miltenyi Biotec), (ii) enzymatic digestions were performed at 35°C, (iii) half the concentration of enzyme P was used, (iv) actinomycin D was used to limit dissociation-induced transcriptional changes, (v) 5% trehalose was added in all buffers to increase cellular viability, (vi) cell clusters were removed by filtration through prewetted 70 μm (352350; Falcon) and 40 μm (352340; Falcon) cell strainers, (vii) erythrocyte and myelin debris removal steps were omitted during dissociation steps, and (viii) all centrifugations were performed at 200 g at 4°C. After dissociation, cells were collected in PBS with 0.2% BSA before being sorted on a Sony SH800 cell sorter. Gates were chosen based on forward and side scatter to exclude myelin debris, erythrocytes, and doublets. Non-viable cells were excluded based on Draq7 and/or DAPI staining (Draq7high and/or DAPIhigh cells were classified as non-viable).
single cells were processed through the Chromium Single Cell Platform using the Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit (v3.1 chemistry, PN-1000121, 10x genomics) and the Chromium Next GEM Chip G kit (PN-1000120) and processed following manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

10x Genomics

Data processing

The alignment to the reference genome, demultiplexing, barcoded processing, gene counting and cell calling were made using the Cell Ranger software (v5.0.0 to v7.00).
Cells were filtered based on output from isOutlier function from scater (v1.20.1), genes expressed in less than two cells were discarded, normalisation was performed by deconvulation ( with scran v1.20.1).
Selection of variable genes, dimensional reduction, batch correction when needed (with fastMNN from batchelor v1.8.0) and clustering (with scran) was performed a first time, then a clusterQC and a stricter QC was performed to retain only high quality cells, annotation was performed with marker genes. More details regarding the differences between datasets can be found in the paper.
Assembly: mm10
Supplementary files format and content: SingleCellExperiment_RDSs contains R files for each dataset. It is the fully cleaned, normalised, batch corrected and annotated version.
Supplementary files format and content: cellinfo_CSVs.tar contains the metadata for the cells from all the cleaned datasets. Each row is a cell and each column has infromation such as the qc parametres or the cell annotation
Supplementary files format and content: cellranger_matrices.tar has the filtered and raw matrices from cellranger output.

Submission date

May 15, 2024

Last update date

May 22, 2024

Contact name

Nadine Bestard-Cuche

E-mail(s)

nadine.bestard@ed.ac.uk

Organization name

University of Edinburgh

Lab

Anna Williams