1. From the provided Master Plate, aliquot Hybridization master mix (5 µl/well) containing a mix of barcoded Reverse GSPs in 1-8 of 96-well plates.
  2. For transferring cells into plates, you have two options: (1) Flow sort T or B cells in a 96-well plate(s) with aliquoted hybridization buffer for single cells per well or 100 cells/well. (2) Deposit a pool of pure T, B cells, or PBMC by adjusting the concentration of the pool to 100 cells/µl and measuring by a hemocytometer.
  3. After hybridization of cellular mRNA with Rev GSPs, non-hybridized Rev GSPs are removed by nuclease treatment, and 96 mRNA-Rev GSP hybrids (each with a cell-specific barcode) are pooled together and purified by SPRIselect magnetic beads.
  4. Pooled, purified mRNA-Rev GSP hybrids are eluted from SPRIselect beads, and cDNA is synthesized by Reverse transcriptase from Rev GSPs.
  5. Sense DNA strand (a complementary strand of cDNA product) is synthesized by DNA polymerase using a mix of Forward (Fwd) GSPs. Fwd GSPs and Rev GSPs are designed to cover both full-length (CDR1-2-3) immune receptor chains and 100-250 bp gene-specific cDNA fragments of T/B cell marker genes (for DriverMap scAIR Immunophenotyping Assay).
  6. Fwd GSP-extended DNAs are amplified using universal anchor primers (AP1 and AP2). The Second round of nested PCR with Illumina’s UD dual-indexed anchor primers allows combining different NGS libraries together (e.g., from two to eight 96-well plates) and adds P5 and P7 sequences necessary for Illumina’s Paired-End 600-nt sequencing on NextSeq 2000/MiSeq instruments.
Last modified: 7 July 2025

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