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Sample GSM7720294

Query DataSets for GSM7720294

Status

Public on Nov 05, 2023

Title

gdTcells, HTO-derived library, VDJ5PGD_HASH

Sample type

SRA

Source name

Liver, Lung, Small Intestine, Lymph nodes, and Spleen

Organism

Mus musculus

Characteristics

tissue: Liver, Lung, Small Intestine, Lymph nodes, and Spleen
cell type: gamma delta T cells
library type: HTO
antibodies/tags: TotalSeq™-C0301 anti-mouse Hashtag 1 Antibody for Spleen, TotalSeq™-C0302 anti-mouse Hashtag 2 Antibody for Lymph nodes, TotalSeq™-C0303 anti-mouse Hashtag 3 Antibody for Liver, TotalSeq™-C0304 anti-mouse Hashtag 4 Antibody for Lung and TotalSeq™-C0305 anti-mouse Hashtag 5 Antibody for Small Intestine

Extracted molecule

protein

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.
Simultaneous single-cell RNA and TCR sequencing was performed using 10x Genomics V(D)J workflow with feature barcoding technology to multiplex cell samples from different organs. Hashtag oligos were obtained as purified and already oligo-conjugated in TotalSeq-C (5’ chemistry) format from BioLegend. Cells were stained with barcoded antibodies together with the staining solution prior to FACS sorting as described above. The antibody concentrations used were 1 µg per million cells, as recommended by the manufacturer (BioLegend) for flow cytometry applications. 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. After the final wash, the cells were resuspended in PBS, filtered through 40 µm cell strainers and processed for sorting. Single-cell TCR libraries were generated using the following primers. 1st PCR: 2 μM of forward primer (5′- GATCTACACTCTTTCCCTACACGACGC-3′) and 0.5 μM of each reverse primer (5′-TCGAATCTCCATACTGACCAAGCTTGAC-3′, 5′-GTCTTCAGCGTATCCCCTTCCTGG-3′, 5′-CTTTCAGGCACAGTAAGCCAGC-3′ and 5′-TCTTCAGTCACCGTCAGCCAACTAA-3′). 2nd PCR: 1 μM of forward primer (5′- GATCTACACTCTTTCCCTACACGACGC-3′) and 1 μM of each reverse primer (5′- CCACAATCTTCTTGGATGATCTGAGACT-3′ and 5′- GTCCCAGTCTTATGGAGATTTGTTTCAGC-3′). 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

RNA-Seq

Library source

other

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Cell Hashing library: read1 file contains cell barcode and UMI; read2 file contains Hashtag Antibody Derived Tag reads
Hashtag-derived oligonucleotide (HTO)

Data processing

Pre-processing of sequencing results to generate count matrices (gene expression, chromatin accessibility, ADT and HTO barcode counts) and TCR clonotypes was performed using the 10x genomics Cell Ranger pipeline.
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). TCR analysis was performed using scRepertoire.
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, filtered_contig_annotations.csv

Submission date

Aug 21, 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