GSM5462420: scRNA-Seq ALS4 1 to 3 and Controls 1 to 3 (Antibody derived tags); Homo sapiens; OTHER
From each patient, blood was drawn into a Cell Preparation Tube with sodium heparin (BD Vacutainer). Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll (Ficoll Hystopaque; Sigma) according to the manufacturer's protocol. PBMCs were aliquoted and frozen at -80°C in FBS (Gibco) containing 10% DMSO (sodium dodecyl sulfate, Sigma). Frozen vials of PBMCs were thawed and washed with culture medium (i.e. RPMI supplemented with 10% FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, and 1 nM sodium pyruvate) containing 2 mg/ml of DNAse I (Roche) (45). Cells were resuspended at the density of 2×106/ml in medium containing 0.1 mg/ml of DNAse I and left in culture for 18 hours in an incubator at 37ºC and 5% CO2 before analysis. PBMCs were incubated on ice for 20 min with 100 ul of the hash mixture in PBS 2% Bovine Serum Albumin (BSA, SIGMA) and then washed three time before CD8 T cell negative selection. human CD8 T cells were purified by negative selection  using a Dynabeads Untouched Human CD8 T Cells Kit (Invitrogen), according to the manufacturer's protocol.Hashed cells were counted on a Nexcelom Autocounter 2000 using AO/PI dye and pooled based on live cell counts. Pooled samples were centrifuged then subsequently resuspended in 100 l of BioLegend's Total Seq C CITE-Seq antibody panel and incubated on ice for 30 minutes. Antibodies were used at a concentration of 1:100 and diluted in wash buffer (PBS, 0.5% BSA, 1mM EDTA). After 3 washes, cells were resuspended in loading buffer (PBS, 0.5% BSA) and counted. Cells were loaded on one lane of the 10x 5'v1.1 assay with a targeted cell recovery of 20,000 cells.The following antibodies were used for CITE-seq and purchased from Biolegend:  TotalSeq-C0046 anti-human CD8   TotalSeq-C0049 anti-human CD3   TotalSeq-C0063 anti-human CD45RA   TotalSeq-C0148 anti-human CD197 (CCR7)   TotalSeq-C0390 anti-human CD127 (IL-7Rα)   TotalSeq-C0088 anti-human CD279 (PD-1)   TotalSeq-C0154 anti-human CD27   TotalSeq-C0386 anti-human CD28   TotalSeq-C0140 anti-human CD183 (CXCR3)   TotalSeq-C0071 anti-human CD194 (CCR4)   TotalSeq-C0141 anti-human CD195 (CCR5)   TotalSeq-C0143 anti-human CD196 (CCR6)   TotalSeq-C0160 anti-human CD1c   TotalSeq-C0085 anti-human CD25   TotalSeq-C0171 anti-human/mouse/rat CD278 (ICOS)   TotalSeq-C0410 anti-human CD38   TotalSeq-C0087 anti-human CD45RO   TotalSeq-C0168 anti-human CD57   TotalSeq-C0159 anti-human HLA-DR   TotalSeq-C0089 anti-human TIGIT (VSTM3)   TotalSeq-C0250 anti-mouse/human KLRG1 (MAFA For scRNA seq and scTCR seq, base call files from a NovaSeq6000 Sequencer (Illumina) and a NextSeq550 Sequencer (Illumina), respectively, were generated by the Human Immune Monitoring Center and the Genomic CoRE at the ISMMS.  BCL files were base-called and demultiplexed using cellranger mkfastq v3.1.0. Alignment of gene expression libraries to human reference GRCh38 as well as the counting of unique features of CITESeq libraries were performed using cellranger count v3.1.0.