Robust RNA Extraction Using Trizol

RNA extraction was performed with Trizol, which proved to ensure good RNA integrity (RIN, 28S/18S ratio) as well as the absence of genomic DNA and contamination [57 (link)]. Briefly, we carefully scraped PDLF off the plates in 1 mL PBS using a cell scraper. Cells were centrifuged at 2000 rpm for 10 min at 4 °C (HERAEUS Fresco 17 Centrifuge, Thermo Fisher Scientific, Waltham, MA, USA). The pellet was reconstituted in 500 µL peqGOLD TriFastTM (30-2010, VWR International, Radnor, PA, USA). After addition of 100 µL chloroform (1.02445.1000, VWR International, Radnor, PA, USA), samples were mixed thoroughly for a minimum of 30 s. Samples were incubated on ice for 15 min and then centrifuged for 15 min at 13,000 rpm and 4 °C. The colourless supernatant was transferred to cold 500 µL isopropanol and mixed. Samples were stored at −80 °C overnight. They were centrifuged for 30 min at 13,000 rpm at 4 °C and the pellet was washed twice with 80% ethanol in RNAse-free water (T143, Carl Roth). After drying the pellet for 30 min, it was reconstituted in 20 µL RNAse-free water (T143, Carl Roth). RNA concentration was determined using a NanoDrop photometer at 260nm (Implen, Munich, Germany).