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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 -

E-mail(s)

sagar@uniklinik-freiburg.de

Organization name

University Medical Center Freiburg

Department

Department of Internal Medicine II