shRNA library screening assay to identify new regulators of hematopoietic cell migration. (A) The murine 32D cell line was transduced with library containing (LTC) or control (CTC) vector particles. shRNA library transduced and control transduced cells were selected based on the migratory capacity (Ma1). The nonmigrating cells were selected and used for migration on 18 consecutive days (MaN). shRNAs were identified in expanded single cell clones of the nonmigrated LTC by standard sequencing. A cultured-only LTC population was used as control. (B) After 18 rounds of migration, the population of nonmigrating LTC had increased to 96%. CTC demonstrated a constant high migration rate, resulting in too few remaining nonmigrated cells to continue the assay. (C) Although no significant difference in migration was observed between LTC and CTC during the first 5 migration cycles, the percent of nonmigrating cells significantly increased in the LTC during migration cycles 6 to 10. (D) CTC and LTC showed no significant difference in cell proliferation during migration cycles 1 to 5. During the next 5 migration cycles, the proliferation rate of CTC increased compared with LTC. *P < .05, ***P < .001. ns, not significant.