GSM7988810: Pooled CD3+ T Cells, scRNAseq; Homo sapiens; RNA-Seq
The microwell-seq barcoding technology (BD Rhapsody platform) was utilized for single cell transcriptomics analysis. Cell viability was determined using trypan blue on the ThermoFisher Scientific, Countess® II FL Automated Cell Counter. Approximately, 0.1X106 T cells were labelled using BD™ Single-Cell Multiplexing Kit-Human with unique tags for each sample and BD™ AbSeq Ab-Oligos as per the manufacturer's guideline (Doc ID: 214419 Rev. 2.0). Subsequently, 20,000 cells (>90% viability) were loaded onto primed microwells of BD Rhapsody cartridge on BD Rhapsody express single-cell analysis system and oligonucleotide barcoded beads were added as per manufacturer's instructions (Doc ID: 210967 Rev. 1.0). Following cell and bead pairing in the microwells, cells were lysed and cDNA was prepared by reverse transcription from poly-adenylated RNA, captured on the beads. The cDNA of mRNA and AbSeq targets was first encoded on BD Rhapsody™ Cell Capture Beads, as described in the instrument user guides. At the same time, the barcode information from BD Rhapsody Cell Capture Beads was also added to AbOligos during reverse transcription, which enables amplification of the AbOligos and Sample Tags in solution. To generate the AbSeq and Sample Tag sequencing libraries, the extended AbOligos and Sample Tags were first denatured from the BD Rhapsody Cell Capture Beads, which were later amplified separately through a series of PCR steps. Meanwhile, the whole transcriptome amplification library was generated directly from the BD Rhapsody Cell Capture Beads using a random priming approach, followed by an index PCR step. The whole transcriptome mRNA, AbSeq, and Sample Tag libraries was combined together for sequencing on Illumina Novaseq 6000 instrument to generate 150bp paired-end reads.