RNAseq of HEK293T cells transfected with a FLAG-N vector
After transfection with Lipofectamine 3000, HEK293T cells were trypsinized and lysed into 700 ul Lysis/Binding Solution (Invitrogen, #AM1912). The lysates were immediately frozen in liquid nitrogen to preserve RNA integrity. Total RNA was then extracted by grinding the frozen cell pellets with pestles and subsequently centrifuging at 13,000 rpm for 3 min at 4C. The supernatant was processed using the RNAqueous Total RNA Isolation Kit (Invitrogen, #AM1912) according to the manufacturer's instructions. RNA quantities were assessed using a Nanodrop ND-1000 Spectrophotometer. Samples were normalized to a concentration of 50 ng/l and submitted to the University of Texas at Austin Genomic Sequencing and Analysis Facility for TagSeq library preparation. Libraries were sequenced on a NovaSeq6000 platform using the SR100 sequencing mode to generate reads. Replicate lanes of each sample were concatenated to produce the final fastqs presented here.