GSM8061464: H_8_8+I_(3) ; HEK293T_8h_10uM-A8+Isrib(biol. rep 3); Homo sapiens; RNA-Seq
Cells were collected with 0.25% trypsin, neutralized with cell culture media, and pelleted at 200 x g for 5 min. Total RNA was extracted using the Quick-RNA MiniPrepKit (Zymo Research), following the protocol suggested by the manufacturer. Libraries were constructed by BGI using their standard methodology. mRNA was enriched from total RNA samples via their polyA tails using oligo dT beads. mRNA was then fragmented and converted to cDNA using random primers to synthesize the first cDNA strand. The second cDNA strand was synthesized using dUTP instead of dTTP. The cDNA was subjected to end-repair and 3' adenylated. Adaptors were ligated to the ends of these cDNA fragments. The U-labeled second-strand template was digested with uracil-DNA-glycosylase. The remaining cDNA fragments were then amplified by PCR. The library was quality controlled, then circularized and amplified to make a DNA nanoball (DNB). Finally, the library was sequenced using the DNBSEQ platform.