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Sample GSM7951843 Query DataSets for GSM7951843
Status Public on Jan 01, 2024
Title BM_2_TCR
Sample type SRA
 
Source name Bone marrow
Organism Mus musculus
Characteristics tissue: Bone marrow
strain: C57BL/6J
Sex: Female
Extracted molecule total RNA
Extraction protocol Bone marrow, lymph node and peripheral blood cells isolated from C57BL/6J female mice were stained with Live/Dead dye and lymphocytes were isolated using a BDFACSAria™ IIISorter. Cells were manually counted by Trypan blue and AO-PI after each centrifugation and resuspension.
By using Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics, 1000263) and Chromium Next GEM Chip K Single Cell Kit (10x Genomics, 1000287), we performed single cell TCR/BCR-seq and 5’ gene expression profiling. The cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Cell-barcoded 5’ gene expression libraries and V(D)J enriched TCR/BCR libraries were sequenced on an Illumina NovaSeq6000 system.
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Data processing Sequencing reads from gene expression and V(D)J libraries were aligned to mm10 mouse reference genome using cellranger 7.0.0 with “multi” mode. The generated count matrices and V(D)J contig annotations were used for downstream analysis performed with Seurat(v4.1.1) and Scirpy(v0.11.2). All samples were aggregated into a single SeuratObject. Cells with gene number (<300 & >97.5% quantile) or high mitochondrial transcript ratio (>25%), and genes expressed in less than 3 cells were excluded. After removing unwanted cells from the dataset, all samples were combined with function "merge". Next, we employed a global-scaling normalization method "LogNormalize" to normalize the feature expression measurements (UMI counts) for each cell by the total expression, then data integration was performed by canonical correlation analysis according to shared sources of variation across multiple datasets using SelectIntegrationFeatures, FindIntegrationAnchors and IntegrateData functions. Highly variable genes (top 3000) were extracted to perform the principal component analysis (PCA) and top 30 of significant principle components were used for cluster analysis. Clusters were visualized using the Uniform Manifold Approximation and Projection (UMAP). Marker genes for each cluster and subgroup were identified by contrasting gene expression of cells from certain cluster or subgroup to that of others using the Seurat FindMarkers function.
Assembly: mm10
Supplementary files format and content: Matrix table with raw gene counts
 
Submission date Dec 07, 2023
Last update date Jan 01, 2024
Contact name yifan zhang
E-mail(s) 23110700099@m.fudan.edu.cn
Organization name fudan university
Street address no
City shanghai
ZIP/Postal code 201508
Country China
 
Platform ID GPL24247
Series (1)
GSE249618 A biphenotypic lymphocyte subset displays both T- and B-cell functionalities
Relations
BioSample SAMN38718429
SRA SRX22819443

Supplementary file Size Download File type/resource
GSM7951843_BM_2_TCR_filtered_contig_annotations.csv.gz 183.9 Kb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA

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