GSM7720294: gdTcells, HTO-derived library, VDJ5PGD_HASH; Mus musculus; RNA-Seq
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.