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Sample GSM6923624

Query DataSets for GSM6923624

Status

Public on Nov 05, 2023

Title

gdTcells, mRNA-derived library parabiosis_resident_p2_2_2_GEX

Sample type

SRA

Source name

Lung, Liver, Spleen, Skin, Lymph nodes and Large Intestine

Organism

Mus musculus

Characteristics

tissue: Lung, Liver, Spleen, Skin, Lymph nodes and Large Intestine
cell type: gamma delta T cells
library type: mRNA

Extracted molecule

polyA RNA

Extraction protocol

Living TCRgd+ single gd T cells from different organs were baroded using hashtag oligos and were FACS sorted in BSA-coated tubes containing 50 µl of PBS. Using pulse geometry gates (FSC‐W × FSC‐H and SSC‐W × SSC‐H), doublets/multiplets will be excluded. After the completion of sorting, the cells will be processed through the different 10x Genomics workflows.
Single-cell RNA sequencing was performed using 10x Genomics with feature barcoding technology to multiplex cell samples from different organs so that they can be loaded on one well to reduce costs and minimize technical variability. Hashtag oligos were obtained as purified and already oligo-conjugated in TotalSeq-B (3’ chemistry) and TotalSeq-C (5’ chemistry) formats from BioLegend. Cells were stained with barcoded antibodies together with the staining solution prior to FACS sorting. Sorted cells were processed through 10x Genomics single-cell 3’ or 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.
To profile gd T cells using antibodies for the quantification of cell surface proteins on a single-cell level, following antibodies were obtained as purified, oligo-conjugated TotalSeq-B reagents from BioLegend - CCR6, NKp46, CD117, KLRG1, CCR7, CD8a, CD5, CD122 (IL-2Rβ), CD127 (IL-7Rα), CD278 (ICOS), Ly-6A/E (Sca-1), CD69, CD44, CD27, CD24, CD62L, CD25, NK-1.1, CD4, CCR2, TCR Vg2, TCRb, CD11c, CD19, GR-1 and CSF1R. Cells were stained with barcoded antibodies together with the staining solution prior to FACS sorting as described above. Antibody concentrations were again 1 µg per million cells, as recommended by the manufacturer (BioLegend) for flow cytometry applications. Sorted gd T cells were processed through 10x Genomics 3’ workflow according to the manufacturer’s instructions. Libraries were pooled in desired ratio together with the gene expression libraries to obtain appropriate sequencing depths as recommended by 10x Genomics and sequenced on a NovaSeq 6000 flow cell.
To perform simultaneous measurement of chromatin accessibility and gene expression, we could not barcode different organs with hashtag oligos and hence, after sorting gd T cells from different tissues, we pooled them to obtain enough cells to perform the nuclei extraction according to 10x Genomics protocol. Thereafter, single nuclei were processed through Chromium Single Cell Multiome ATAC + Gene Expression workflow and processed according to the manufacturer’s instructions. Gene expression and chromatin accessibility libraries were sequenced to obtain appropriate sequencing depths as recommended by 10x Genomics using Illumina NovaSeq 6000 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

gdTcells, mRNA-derived library
antibodies/tags: none
mRNA library: read1 file contains cell barcode and UMI; read2 file contains transcript
poly A RNA

Data processing

Pre-processing of sequencing results to generate count matrices (gene expression, chromatin accessibility, ADT and HTO barcode counts) was performed using the 10x genomics Cell Ranger pipeline where ADT and HTO sequences were concatenated.
Further processing was done with Seurat and Signac (cell and gene filtering, hashtag identification, clustering, differential gene expression analysis based on gene expression as well as chromatin accessibility).
Assembly: Alignments were performed using pre-build Cell Ranger and Cell Ranger ARC mouse mm10 references.
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz, filtered_feature_bc_matrix.h5, atac_fragments.tsv.gz

Submission date

Jan 09, 2023

Last update date

Nov 05, 2023

Contact name

Sagar -

E-mail(s)

sagar@uniklinik-freiburg.de

Organization name

University Medical Center Freiburg

Department

Department of Internal Medicine II