GSM5004409: GEX_TCR-95-BL; Homo sapiens; RNA-Seq
The cells were first counted and measured for viability using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter) and a basic hemocytometer and light microscope.  Barcoded Gel Beads were thawed from -80 oC to room temperature and the cDNA master mix was prepared according to the manufacture's instruction for Chromium Single Cell 3' v2 library kit (10x Genomics).  The target capture number of cells was 2000-3000, so a volume containing more than 5000 live cells was combined with cDNA master mix.  The cell suspension and master mix, thawed Gel Beads and partitioning oil were added to a Chromium Single Cell A chip.  The filled chip was loaded into the Chromium Controller, where each sample was processed and the individual cells within the sample were captured into uniquely labeled GEMs (Gel Beads-In-Emulsion).  The GEMs were collected from the chip and taken to the bench for reverse transcription, GEM dissolution, and cDNA clean-up. The resulting cDNA was a pool of uniquely barcoded molecules.  Cleaned and measured pooled cDNA continued on to library construction, where standard Illumina sequencing primers and a unique i7 Sample index were added to each cDNA pool. All cDNA pools and resulting libraries were measured using Qubit High Sensitivity assays (Thermo Fisher Scientific), Agilent Bioanalyzer High Sensitivity chips (Agilent) and Kapa DNA Quantification reagents (Kapa Biosystems).   Libraries were sequenced at 60,000 fragment reads per cell following Illumina's standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit.  The flow cells were sequenced as 100 x 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software.  Base-calling was performed using Illumina's RTA version 2.7.3.