Mkfastq (Cell Ranger 3.1.0) was used to generate FASTQ files with default parametersĬount (Cell Ranger 3.1.0) was used to generate the filtered UMI matrix with default parameters Single-cell RNA-seq with 10x Genomics 5' paired-end Libraries were constructed using Chromium™ Single Cell 5' v1 Reagent Kits following the manufacturer’s protocolġ00 x 100 bp paired-end reads, 3'TAG, Hg19 human reference genome. Single-cell RNA-seq using 10x genomics platform was performed using Chromium™ Single Cell 5' v1 Reagent Kits following the manufacturer’s protocol Following careful mixing, 33 µl of the cell suspension was transferred to a PCR-tube for processing as per the manufacturer’s instructions (10x Genomics). 25 µl of resuspension buffer (0.22 µm filtered ice-cold PBS supplemented with ultra-pure bovine serum albumin 0.04 %, Sigma-Aldrich) was added to the tube and the pellet was gently but thoroughly resuspended. Cells were then centrifuged for 5 minutes (600 g at 4 ˚C) and the supernatant was carefully removed leaving 5 to 10 µl. Following sorting, ice-cold PBS was added to make up to a volume of 1400 µl. Following elution, enriched populations were immediately sorted using a FACSAria Fusion Cell Sorter (Becton Dickinson) based on dual expression of CD154 and CD69 for 6-hour stimulation condition, and CD137 and CD69 for 24-hour stimulation condition.įor combined single-cell RNA-seq and TCR-seq assays (10x Genomics), a maximum of 60,000 virus-reactive memory CD4+ T cells from up to 8 donors were pooled by sorting into low retention 1.5mL collection tubes, containing 500 µL of a1:1 solution of PBS:FBS supplemented with RNAse inhibitor (1:100). The flow-through from the column was collected and re-plated for harvesting cells responding 24 h after peptide stimulation.Īnalogous to enrichment for CD154+, CD137-expressing CD4+ memory T cells cells were positively selected by staining with biotin-conjugated CD137 antibody (clone REA765 Miltenyi Biotec) followed by anti-biotin MicroBeads and applied to a new MS column. Labelled cells were added to MS columns (Miltenyi Biotec) and positively selected cells (CD154+) were eluted and used for FACS sorting of CD154+ memory CD4+ T cells. For subsequent MACS-based enrichment of CD154+, cells were sequentially stained with fluorescence-labeled surface antibodies, Cell-hashtag TotalSeq™-C antibody (0.5 µg/condition), and a biotin-conjugated CD154 antibody (clone 5C8 Miltenyi Biotec) followed by anti-biotin microbeads (Miltenyi Biotec). Cells were stimulated by the addition of individual virus-specific peptide polls (1 µg/ml) for 6 h in the presence of a blocking CD40 antibody (1 µg/ml Miltenyi Biotec). PBMCs from each donor, were thawed, washed, plated in 6-well culture plates at a concentration of 5 x 106 cells/ml in 1 ml of serum-free TexMACS medium (Miltenyi Biotec) and left overnight (5 % CO2, 37 ˚C). GEO help: Mouse over screen elements for information.Ĭell type: Memory CD4 T cells, helper enriched: CD3+ CD4+ CD45RA- CD154+ CD69+
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