GSM5381094: Niclosamide Vero E6 treated samples at 48 hours rep2; Chlorocebus sabaeus; RNA-Seq
Cell pellets were resuspended in 1mL Trizol, 200 µL chloroform was added to each sample, and samples were mixed thoroughly by vortexing, then incubated at room temperature for 3 minutes. Samples were centrifuged at 12,000xg for 15 minutes at 4°C. The upper aqueous layer was transferred to a new tube and 500 µL isopropanol was added and mixed by vortex-ing, prior to incubation at room temperature for 10 minutes. RNA was pelleted by centrifuging at 12,000 xg for 10 minutes at 4°C, and supernatant was discarded. The pellet was rinsed in 75% ethanol and pelleted by centrifuging at 7,500xg for 5 minutes at 4°C. Ethanol wash and spin were repeated a second time. The pellet was air dried in an inverted tube. Genomic DNA was digested using the TURBO DNA-free™ Kit (Invitrogen #AM1907) according to manufac-turer instructions. Samples were mixed with 350 µL buffer RLT from the RNeasy® Mini kit (Qi-agen #74104) and 250 µL 100% ethanol, then transferred to a RNeasy® Mini kit spin column. Columns were centrifuged at 8,000xg for 15 seconds at room temperature, and flow-through was discarded. Buffer RPE (500 µL) was added to the column, which was centrifuged at the same conditions, and flow-through was discarded. The RPE wash was repeated a second time. The column was transferred to a fresh collection tube and centrifuged at 12,000 xg for 1 min to dry. RNA was eluted by adding 50 µL of nuclease free water and centrifuging for 1 mi-nute at 8,000xg. RNA libraries were prepared for sequencing using standard Illumina protocols