RNA-Seq of HEK293FT cells transfected with RADARs
HEK293FT cells were maintained in D10 media (DMEM supplemented with 10%FBS) and passaged every 2-3 days before reaching confluency. Approximately 10k cells were plated per well of 96 well dish the day prior to infection. Cells were transfected with RADARS plasmids as follows: 20ng of ADAR plasmid, 20ng of sensor, and 20ng of targets were delivered to each well. For controls, relevant plasmids were replaced with pUC19. The cells were allowed to grow for 2days post transfection before harvesting. Total RNA was extracted using the RNeasy 96 Kit (Qiagen) with DNase treatment. 5 uL of eluant (~250 ng) was taken as starting material for transcriptome-wide sequencing prep. Libraries were prepared using the KAPA mRNA HyperPrep Kit (KAPA Biosystems) according to manufacturer instructions. The mRNA fragmentation step was carried out for 6 minutes at 85 deg C. KAPA Unique Dual-Indexed Adapters diluted to 1.5 uM were used in the adapter ligation step and PCRd with Illumina primer mix supplied in Kapa kit. Libraries were diluted to a final concentration of 0.5 nM, pooled, and sequenced on an Illumina NextSeq 500 (75 cycles, paired end reads)