SEM cell line, USO1 knock down, replicate 2
Lentiviral preparation expressing guide RNA 3 to target USO1 was used to transduce SEM cells expressing Cas9 alongwith 1L of 1mg/mL stock. The cells were spin infected at 2500 rpm for 90 mins. at 30 degree celcius. Transduced cells were resuspended in growth media and cultured for 48 hrs prior to FACS sorting. Cells were sorted based on GFP+ tRFP+ double positive population and expanded in culture post sorting. The knockdown of USO1 was confirmed by RT-qPCR and by Western blotting technique. Once the knockdown is confirmed, RNA was isolated from both NT and USO1 depleted cells and submitted for RNA sequencing.