Sample 51-TCR_Pan_T8010353_p
Briefly, solid tissues were transferred to a 100mm tissue culture dish, cut into small pieces and transferred to C-tubes (Miltenyi Biotec) at a maximum of 5g/tube in 5mL of X-vivo15 media containing 0.13U/m Liberase TL (Roche), 10U/mL DNase (benzonase nuclease, Millipore/Merck) supplemented with 2% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco), penicillin (100 U/ml, Sigma-Aldrich), streptomycin (0.1 mg/ml, Sigma-Aldrich), and 10mM HEPES (Sigma Aldrich). The samples were then dissociated using a GentleMACS Octo dissociator (Miltenyi Biotec) using a protocol that provided gradual ramping up of homogenisation speed along with 2x 15 minute heating/mixing steps at 37°C. Resulting single cell suspension was layered over ficoll and spun at 400g for 20 minutes, with no brake.  The interface layer containing the MNCs were collected and transferred to a fresh tube and washed in PBS+BSA. Blood and bone marrow MNC were washed with PBS and exposed to the same enzymatic conditions as solid tissues by resuspending cell pellets in tissue dissociation media containing liberase TL for 30 minutes at 37°C prior to counting and resuspending in 10X buffer. This protocol applies to donors A29, A31, A35, A36, A37, A52  Following the manufacturer's instructions for Chromium Single Cell 5' V(D)J Reagent Kits (v1 Chemistry)