RNA-Seq of activated 293T cell
Forty-eight hours post-transfection, cells were pelleted and washed with DPBS. Total RNA and DNA were extracted. All mRNAs were subjected to a reverse-transcription reaction in 8-strip PCR tubes. The purified cDNA was split into 8 PCR tubes and amplified using NEBNext Ultra II Q5 Master Mix and F-sg9-rd1 and P7-i7 (index)-rd2 primers for eight cycles. After the PCR reaction, all eight tubes for each sample were combined in a DNA LoBind tube. PCR products were purified using AMPure XP (Beckman Coulter, A63881) and then further amplified using P5-i5 (index)-rd1 and P7 primers for fifteen PCR cycles to add P5, P7, and unique 8 bp Illumina index sequences.