GSM9169475: HEK293 transfected, 18 h after transfection, rep 3; Homo sapiens; RNA-Seq
For total RNA isolation, 1E7 cells were harvested at 18 h after transient transfection and centrifuged at 800 x g and 4 °C for 7 min. The pellet was lysed in 1 mL of TRI Reagent (Zymo Research) followed by addition of 200 µL of chloroform. The mixture was vortexed for 15 s, incubated at room temperature for 3 min, and centrifuged at 12,000 x g at 4 °C for 15 min. To precipitate the RNA, isopropanol was added (1:1) and the tube was inverted 3 times. After an incubation of 10 min at room temperature, the mixture was centrifuged at 12,000 x g at 4 °C for 10 min. The RNA pellet was washed twice with ethanol (75%) and centrifuged at 7,500 x g at 4 °C for 5 min each time. The ethanol was discarded, and the pellet was dried for 10 min. The RNA was solubilized in 50 µL of RNase-free water. The concentration was determined using a NanoDrop One (Thermo Fisher Scientific). A 75 nt paired-end, stranded sequencing approach was performed on a NextSeq system (Illumina). RNA libraries for RNA-seq were prepared using a TruSeq Stranded Total RNA kit (Illumina) following manufacturer's protocols.