Invitrogen geneart platinum cas9 nuclease

To knock out HDAC7 gene expression in RPMI-8226wt cells, we designed six guide RNAs (gRNAs) specific to a sequence in the HDAC7 region. The RMPI-8226wt cells were transiently electroporated with 240 ng of each gRNA mixed with 500 ng Invitrogen GeneArt™ Platinum™ Cas9 Nuclease (Invitrogen Carlsbad, CA) using the Invitrogen Neon Transfection System (Invitrogen, Carlsbad, CA) at 1100 v, 30 ms, and 2 pulses. Subsequently, protein lysates harvested from cells with each of the six gRNAs were immunoblotted for HDAC7 to identify the gRNA that provided the most decrease of HDAC7 gene expression (5’-AAACCCCCTGGATGCACAGCCCCGGCG-3’) among the 6 gRNA. Afterward, cells with the above-mentioned gRNA were sorted as single cells in CoSTAR ultra-low cluster, 96-well plates (Corning Inc, Corning, NY), with conditioned media to allow single-cell cloning and expansion. This process was repeated once to identify sub-clones #1 and #2 with sufficient knockdown of HDAC7 gene expression.