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Sample GSM7221682 Query DataSets for GSM7221682
Status Public on Jun 07, 2023
Title Control_Spleen
Sample type SRA
Source name Spleen
Organism Mus musculus
Characteristics tissue: Spleen
cell type: CD45+ cells
genotype: C57BL/6
treatment: Sham induction
Extracted molecule total RNA
Extraction protocol Single-cell suspensions were prepared from pooled mouse spleen and brain tissues at 72 h after ICH induced by autologous blood injection or sham operations.CD45+ cells were isolated at 4°C using flow-cytometry.As there are few T cells in sham brain, we optimized the FACS-sorting and also isolated the the CD45hi cells in the sham brain. This allows us to obtain enough CD4+ T cells in sham brains for sub-clustering and data processing.
Library was performed according to the manufacter’s instructions (single-cell 3' v.2 kit, 10× Genomics). Cell viability was ≥90% for all samples. Cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel beads-in- emulsion (GEM), which were used to reverse-transcribe mRNA and amplify the derived cDNA. Specifically, gel beads work as delivery system of millions of oligo primers each containing (a) a partial Read 1 primer sequence used for sequencing on the flow cell, (b) a 16-bp cell barcode called 10× barcode that serves as the unique molecular address for that particular bead, (c) the unique molecular identifier (UMI) which is a 10-bp randomer different for each oligo of a bead used for digital mRNA counting, and (d) a 30-bp poly dT tail which allows the primers to hybridize to mature mRNAs for first-strand cDNA synthesis. Once partitioned with the cells and reverse transcription (RT) reagents, the gel bead dissolves and its oligo primers are released into the aqueous environment of the GEM. The cell captured in the GEM is also lysed and the content of the GEM (oligos, lysed cell components, and master mix) is incubated in an RT reaction to generate full-length, barcoded cDNA from the poly A-tailed mRNA transcripts. The RT reaction is primed by the barcoded gel bead oligo and the reverse transcriptase incorporates a template switch oligo via a template switching reaction at the 50 end of the transcript. The GEMs are then broken and single-stranded barcoded cDNAs from thousands of cells are pooled. A bulk cDNA PCR amplification follows to generate enough material for library generation. During library preparation after an enzymatic fragmentation, Read 2 is added by adapter ligation, and finally paired-end constructs with P5 and P7 sequences and a sample index were added.
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model DNBSEQ-G400
Description 10x Genomics
Data processing Raw files (FASTQ format) were processed with Cell Ranger software (v5.0.1), which performed mapping to the mm10 transcriptome, filtering, barcode counting and UMI counting and finally generated feature-by-barcode UMI matrix. Scrublet software was employed to remove potential doublets for individual samples. Seurat (v3.1.5) was used for downstream analysis. Low-quality cells, <500 genes expressed or with <2500 UMIs, were excluded at the initial quality-control step. Cells >15% mitochondrial UMIs were removed. Library-size normalization was performed on the UMI-collapsed gene expression values for each cell barcode, by scaling the total number of transcripts and multiplying by 10,000. Data were then log-transformed for further downstream analysis.
Assembly: mm10
Supplementary files format and content: tab-separated values files
Submission date Apr 24, 2023
Last update date Jun 07, 2023
Contact name Wei-Na Jin
Organization name Beijing Tiantan Hospital, Capital Medical University
Department China National Clinical Research Center for Neurological Diseases
Street address NO.119, Southwest of the Fourth Ring Road, Fengtai District
City Beijing
State/province Beijing
ZIP/Postal code 100070
Country China
Platform ID GPL28457
Series (1)
GSE230414 Molecular signatures of CD45+ cells from brain and spleen after intracerebral hemorrhage
BioSample SAMN34353023
SRA SRX20083022

Supplementary file Size Download File type/resource 20.1 Mb (ftp)(http) TAB
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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