GSM3589417: PP014; Homo sapiens; RNA-Seq
Tissues acquired from donors were maintained in cold saline during transport to the laboratory, typically within 2-4 hours of procurement. We flushed the left lobe of the lungs with cold complete medium (RPMI 1640, 10% FBS, 100 U/ml penicillin, 100 ug/ml streptomycin, 2 mM L-glutamine) and isolated lymph nodes from the hilum, near the intersections of major bronchi and pulmonary veins and arteries, removing all fat. To obtain mononuclear cell suspensions, hilar lymph nodes and the left lateral basal segment of the lung were mechanically processed using a gentleMACS tissue dissociator (Miltenyi Biotec), enzymatically digested (complete medium with 1 mg/ml collagenase D, 1 mg/ml trypsin inhibitor and 0.1 mg/ml DNase for 1 hour at 37°C in a mechanical shaker) and centrifuged on a density gradient using 30% Percoll Plus (GE Healthcare). We aspirated bone marrow from near the anterior superior iliac crest. For bone marrow and peripheral blood, we isolated mononuclear cells by density gradient centrifugation using Lymphocyte Separation Medium (Corning). Next, we enriched single cell suspensions from all tissues and blood for untouched CD3+ T cells using magnetic negative selection (MojoSort Human CD3+ T cell Isolation Kit; BioLegend). To eliminate any dead cells prior to stimulation, we used a dead cell removal kit (Miltenyi Biotec). We cultured 0.5 – 1 x 106 CD3+ enriched cells from each donor tissue for 16 hours at 37°C in complete medium, with or without TCR stimulation using Human CD3/CD28 T Cell Activator (STEMCELL Technologies). After stimulation, we removed dead cells as above before preparing cells for single-cell RNA-seq. We loaded single-cell suspensions into a Chromium Single Cell Chip (10x Genomics) according to the manufacturer's instructions for co-encapsulation with barcoded Gel Beads at a target capture rate of ~5,000 individual cells per sample. We barcoded the captured mRNA during cDNA synthesis and converted the barcoded cDNA into pooled single-cell RNA-seq libraries for Illumina sequencing using the Chromium Single Cell 3' Solution (10x Genomics) according to the manufacturer's instructions. We processed all of the samples for a given donor simultaneously with the Chromium Controller (10x Genomics) and prepared the resulting libraries in parallel in a single batch.  We pooled all of the libraries for a given donor, each of which was barcoded with a unique Illumina sample index, for sequencing in a single Illumina flow cell.