DDX50 shRNA knock down or control shRNA in primary neonatal keratinocytes in differentiation conditions to determine the effect of DDX50 on RNA stability, using actinomycin D.
Primary human keratinocytes were isolated from fresh surgically discarded newborn foreskin, and progenitor cells cultured in complete Keratinocyte-SFM (Life Technologies, #17005-142) and Medium 154 (Life Technologies #M-154-500). Human skin keratinocyte cells were induced to differentiate by the addition of 1.2 mM calcium for 3 days at full confluence in the 50/50 media. Biological replicates were performed in all cases using primary cells from at least 2 independent, unrelated donors. For actinomycin D treatment, differentiated keratinocytes were treated with 5 µg/mL actinomycin D (~4 µM) or DMSO for the indicated time before harvesting the cells. Total RNA was extracted using the RNeasy Plus kit (QIAGEN, 74136), followed by poly(A) enrichment and library preparation using the NEB Ultra II (non-directional) kit (E6111).
NIAMS-phs003766
a_shDDX50-4_0h_biorep1_stability_DDX50