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Sample GSM8016245 Query DataSets for GSM8016245
Status Public on Mar 11, 2024
Title iNKTcells, HTO-derived library
Sample type SRA
Source name Thymus
Organism Mus musculus
Characteristics tissue: Thymus
cell type: Invariant natural killer T cells
genotype: C57BL/6
library type: HTO
antibodies/tags: TotalSeq™-C0301 anti-mouse Hashtag 1 Antibody for Stage 0 iNKT from Ctrl 1 TotalSeq™-C0302 anti-mouse Hashtag 2 Antibody for Stage 3 iNKT from Ctrl 1 TotalSeq™-C0303 anti-mouse Hashtag 3 Antibody for Stage 0 iNKT from Ctrl 2 TotalSeq™-C0304 anti-mouse Hashtag 4 Antibody for Stage 3 iNKT from Ctrl 2 TotalSeq™-C0305 anti-mouse Hashtag 5 Antibody for Stage 0 iNKT from T-KO 1 TotalSeq™-C0306 anti-mouse Hashtag 6 Antibody for Stage 3 iNKT from T-KO 1 TotalSeq™-C0307 anti-mouse Hashtag 7 Antibody for Stage 0 iNKT from T-KO 2 TotalSeq™-C0308 anti-mouse Hashtag 8 Antibody for Stage 3 iNKT from T-KO 2
Extracted molecule protein
Extraction protocol To perform the single-cell RNA sequencing experiments, cells of 4 thymi of Ctrl mice were pooled and the same was done with 4 thymi of T-KO mice. Cells were stained with CD1d tetramers, anti-TCRβ, anti-NK1.1, anti-CD44 and anti-CD24 antibodies on ice. Cells were sorted by flow cytometry for TCRβ+, CD1dt+, CD24+, NK1.1- cells to obtain stage 0 iNKT cells, and for TCRβ+, CD1dt+, NK1.1+, CD24-, CD44+ cells to obtain stage 3 iNKT cells. This was done twice in parallel, to have two replicas each. After sorting, each cell population was barcoded by hash-tagged antibodies (TotalSeqC format, anti-mouse Hashtag 1 to 8; clone M1/42, 30-F11, BioLegend). The antibody concentrations used were 1 ug per million cells. After staining, cells were washed three times in PBS containing 2% BSA and 0.01% Tween 20, followed by centrifugation (300 x g 5 min at 4 °C) and supernatant exchange. Then all 8 populations were pooled (2x stage 0 Ctrl, 2x stage 0 T-KO, 2x stage 3 Ctrl, 2x stage 3 T-KO).
Single-cell RNA sequencing was performed using 10x Genomics with feature barcoding technology to multiplex cell samples from different mice well to reduce costs and minimize technical variability. Hashtag oligos were obtained as purified and already oligo-conjugated in TotalSeq-C (5’ chemistry) formats from BioLegend. Cells were stained with barcoded antibodies after sorting. Cells were processed through 10x Genomics single-cell V(D)J workflow according to the manufacturer’s instructions. Libraries were pooled to desired quantities to obtain appropriate sequencing depths as recommended by 10x Genomics and sequenced on a NovaSeq 6000 flow cell.
Library strategy OTHER
Library source other
Library selection other
Instrument model Illumina NovaSeq 6000
Description Hashtag-derived oligonucleotide (HTO)
Data processing Pre-processing of sequencing results to generate count matrices (gene expression and HTO barcode counts) was performed using the 10x genomics Cell Ranger pipeline.
Further processing was done with Seurat (cell and gene filtering, hashtag identification, clustering, differential gene expression analysis based on gene expression).
Assembly: Alignments were performed using pre-build Cell Ranger mouse mm10 reference.
Supplementary files format and content: 10x Genomics output files are barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz. Stage 0 and Stage 3 NKT datasets were analyzed using Seurat. Seurat R objects are called nkt_stage0_final.rds and nkt_stage3_final.rds
Library strategy: scRNA-seq and CITE-seq
Submission date Jan 13, 2024
Last update date Mar 11, 2024
Contact name Sagar -
Organization name University Medical Center Freiburg
Department Department of Internal Medicine II
Lab Sagar
Street address Hugstetter Straße 55
City Freiburg
ZIP/Postal code 79106
Country Germany
Platform ID GPL24247
Series (1)
GSE253196 Kidins220 and Aiolos promote thymic iNKT cell development by reducing TCR signals
BioSample SAMN39431456
SRA SRX23206017

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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