Single-cell derived parent clones and their kindred single cells were prepared and collected using CellRaft arrays (Cell Microsystems) as described previously (11). Briefly, an LSC suspension was plated on a CellRaft array consisting of 12,000 individual portable rafts for single cells at the required density of 5000 cells per array. After 4 to 8 hours, individual LSCs were elongated and attached to the array surface locating on individual rafts. After attachment, the medium with floating cells was replaced, and single-cell positions were marked and tracked during the following 7 to 10 days to detect dividing cells and growing individual single-cell derived clones. Once the colony/clone reached confluence on the raft (8 to 10 cells per raft), it was dislocated from the array with a positioned automatic needle and transferred with a magnetic wand to a 96-well plate. Upon reaching confluence, single-cell derived clones were trypsinized and subsequently transferred to 24-well plates, then 12-well plates, 6-well plates, and, lastly, 10-cm plates to reach a total amount of 1.5 × 106 to 3 × 106 cells per parent clone. Together, the process of establishing a clone from a single cell took about 25 to 30 days.

Individual single cells from the parent clones were collected, also using CellRafts, and transferred to a 0.2-ml PCR tube containing 2.5 μl of PBS. The presence of a single raft was observed under a magnifying glass. Upon single-cell collection, tubes were fast frozen on dry ice and kept on −80°C until further use.

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