CLIP-seq of HA-tagged DDX50 in primary neonatal keratinocytes under progenitor growth conditions (Prog), low glucose differentiation conditions (L_gluc), or normal high glucose differentiation conditions (H_gluc) to determine the effect of cell state on RNA interactions.
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). Cells were induced to differentiate by the addition of 1.2 mM calcium for 3 days at full confluence. Cells with low glucose and the corresponding normal glucose condition were maintained in glucose-free DMEM (Gibco, #11966025) supplied with SFM supplement (rEGF: Gibco, #10450-013; BPE: 13028-014), 1X Sodium Pyruvate (Gibco, 11360070) and a final concentration of 0.5 g/L (“low” glucose, Gibco, A2494001) or 4.5 g/L glucose (“normal” glucose) with adding 1.2 mM calcium during differentiation for 3 days. All the “low” and “normal” conditions indicated referred to day 3 differentiated cells if not specified. Cells were lysed by lysis buffer and sonication, followed by immunopurification of the protein of interest, according to the easyCLIP protocol (https://doi.org/https://doi.org/10.1002/cpz1.659).
NIAMS-phs003766
a_STAU1_shDDX50-1_1