2 ml tube

Primary and metastatic tissues from biopsy and surgical mass were dissected and transferred to a 2 ml tube (Axygen, China) each containing 1 ml pre-warmed M199-media (ThermoFisher Scientific, USA), 2 mg/ml collagenase P, 2.5 mg collagenase D (Roche, USA) and 10U/μl DNase I (Roche, USA) as was adapted from Tirosh et al.’s protocol [39 (link)]. Tissues were digested for 60 min at 37 °C and then pipetted up and down every 10 times every 10 min. The tissue suspensions were then filtered with a 70 μm nylon mesh (ThermoFisher Scientific, USA) and centrifuged at 450 × g for 5 min. Pellet was resuspended for live cell staining using CFSE incubation for 5 min.

Zein protein, which is hydrophobic, could hinge with starch granules into starch–protein matrix in corn endosperm, thus the zein protein contents in corn determine the rate and extent of starch degradation. An organic solvent was applied to extract zein proteins according to Liu et al. [14 (link)] to represent the starch–protein matrix differences between raw and steam flaked corns. In brief, 100 mg air dry corn samples were mixed with 1 mL of zein extraction buffer (70% [vol/vol] ethanol, 2% [vol/vol] 2-mercaptoethanol, 3.75 mM sodium borate [pH 10], 0.3% SDS) in a 2 mL tube (Axygen), and centrifuged at 15,700 × g for 20 min after 2 h of incubation at room temperature. Then 100 μL of the supernatant, along with an additional 10 μL of 10% [wt/vol] SDS solution, was transferred into a new tube and freeze-dried. Next, 100 μL distilled water was added to dissolve the protein. Finally, a mixture of 8 μL protein solution and 2 μL protein loading buffer was analyzed by SDS-PAGE gel (15% [wt/vol]) for γ-zein accumulation patterns.

Fresh skin biopsy samples including primary cSCC and patient-matched normal skin were minced. Each tissue was dissociated in a 2 ml tube (Axygen, China) containing 1 ml pre-warmed M199-media (ThermoFisher Scientific, USA), 2 mg/ml collagenase P (Roche, USA), and 10 U/μl DNase I (Roche) according to the protocol described by Tirosh et al. [16 (link)]. Tissues were digested for 60 min at 37 °C and then pipetted up and down every 10 min for 10 times. After initial isolation, cells were filtered by a 70 μm nylon mesh (ThermoFisher Scientific) and were spun at 450 × g for 5 min to yield single-cell suspensions. Pellet was resuspended for live cell staining using carboxyfluorescein diacetate succinimidyl ester incubation for 5 min.