GSM8389466: Human donor 2, expanded, scRNAseq; Homo sapiens; RNA-Seq
iNKT scRNA-seq libraries were prepared from both freshly purified iNKTs and matched iNKT cellular products at the end of the expansion. Each library was generated from 10,000 cells with viability >90% as confirmed by Trypan Blue. Human and canine iNKT cells were immunomagnetically purified from PBMCs using PE-conjugated human CD1d tetramers loaded with the αGC analogue PBS57 (NIH Tetramer Core Facility) and anti-PE microbeads (Miltenyi Biotec). After purification, cells were resuspended in PBS with 0.04% w/v BSA for scRNAseq library preparation. For canine and human iNKT expansion, CD1d+ artificail antgen presenting cells were pulsed overnight with αGC 100ng/ml (KRN7000, Cayman Chemicals) and irradiated on the following day (i-aAPC, 10,000 rad). Purified iNKT cells were seeded in round-bottom 96-wells at 1:1 ratio with i-aAPC and maintained at 38-38.8˚C in IMDM medium containing 25 mM Hepes (Gibco), 10% heat inactivated FBS (Atlanta Biologicals), 1% Pen/Strep (Gibco), 1% glutamax (Gibco), 1% non-essential amino acids (Gibco), 1mM sodium pyruvate (Gibco) and 50µM 2-Mercaptoethanol (Gibco). Cross-reactive human IL-15 (Miltenyi Biotec, 30 ng/ml) was supplemented 24-48h after stimulation. Human IL-15 and human IL-2 (Miltenyi Biotec, 30 ng/ml and 100 IU/ml respectively), human IL-7 and human IL-21 (Peprotech, 10 ng/ml) were added 6 days after stimulation and then every other day. To generate clinical scale iNKT products, iNKTs were restimulated twice, i.e., during the second and third week of expansion.8 days after the last restimulation, 10,000 iNKTs were resuspended in PBS with 0.04% w/v BSA for scRNA-seq library preparation. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, iNKT cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.