CLIP-seq of HA-tagged NSUN2 in primary neonatal keratinocytes under progenitor growth conditions (Prog), low glucose differentiation conditions (lowGlc), or normal high glucose differentiation conditions (normGlc) 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).