RNA Extraction from In Vitro and In Vivo Samples

In vitro and in vivo RNA extraction were carried out as described previously (7 (link), 9 (link)). For in vitro RNA extraction, cells were collected at different time points, centrifuged for 2 min at 10 K rpm at room temperature and RNA was extracted according to the manufacturer’s protocol (MasterPure Yeast RNA Purification Kit). For in vivo RNA extraction, mice were euthanized, ear tissue was harvested, and the tissue was placed into the ice-cold RNA-Later solution. Ear tissue was then transferred to the mortar and flash-frozen with liquid nitrogen. Using a pestle, the frozen was ground to a fine powder. The resulting powder was collected and 1 mL of ice-cold Trizol was added. The samples were placed on a rocker at RT for 15 min and then centrifuged at 10K at 4°C for 10 min. The cleared Trizol was collected into a 1.5 mL Eppendorf tube and 200 µL of RNase-free chloroform was added to each sample. The tubes were shaken vigorously for 15 s and kept at RT for 5 min followed by centrifuge at 12 K rpm at 4°C for 15 min. The cleared aqueous layer was then collected into a new 1.5 mL Eppendorf tube and RNA was further extracted following the Qiagen RNeasy kit protocol.