GSM4746102: 22RV1_SLAM_DMSO_2h_2; Homo sapiens; RNA-Seq
22RV1 carcinoma cells were seeded at 60% confluency 18h before being pretreated with 800uM s4U (Sigma, St. Louis, MO) to start the pulse. The cells were incubated in the media with s4U and the 0h samples were collected after 30 minutes by washing the cells with PBS and lysing directly with TRIzol (Invitrogen, Carlsbad, California). The media was replaced with media containing 800uM s4U along with either DMSO, KB742 (1.2uM) or KI-ARv-03 (4uM) and they were collected similarly after 2,4 or 8h post incubation. The RNA from the cells were harvested by total RNA extraction in the presence of 0.1mM DTT to maintain the samples in reducing conditions and the extracted RNA was resuspended with 1mM DTT.  The ERCC RNA Spike-in control mix was added to each sample and thiol modifications were performed by treating 1.5ug of total RNA with freshly prepared Iodoacetamide (IAA) at a final concentration of 10mM. The reactions were quenched, and the thiol modified RNA was ethanol precipitated The libraries were prepared using the QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) and were sequenced with NextSeq 500/550 High Output Kit v2.5 (75 Cycles) (Illumina, San Diego, CA).