GSM5618376: Single cells on plate LCE513 indexed with RPI-9; Homo sapiens; RNA-Seq
Neonatal and paediatric thymus and blood samples were taken from consented cardiac surgery patients (HREC38192) and obtained from the Melbourne Children's Heart Tissue Bank (MCHTB) for this analysis. Peripheral blood mononuclear cells were isolated by a Ficoll-Paque density gradient centrifugation (supplier). Donor thymi were cut into small piece and passed through a 70mM into PBS containing 2% fetal calf serum (FACS buffer and washed x 2), prior to magnetic bead enrichment. Single-cell suspensions of human thymus were enriched for Vg9Vd2+ T cells by staining for Vg9-BV421 or Vg9-APC antibody followed by Vd2-PECy7 antibody or Vd2-PE antibody. Magnetic bead enrichment proceeded using anti-PE microbeads and passage over LS columns as per manufacturer's instructions (Miltenyl Biotec). Single cell transcriptome libraries were generated using the CelSeq2 protocol (Hashimshony et al., 2016 Genome Biol.) with the following adaptations: Second strand synthesis was performed using NEBNext Second Strand Synthesis module (NEB, #E6111S) in a final reaction volume of 8 µl and NucleoMag NGS Clean-up and Size select magnetic beads (Macherey-Nagel, 7449970.5) were used for all DNA purification and size selection steps.