GSM3416118: LD1rep3; Homo sapiens; RNA-Seq
Twenty hours post-exposure to X-ray radiation or negative control conditions, 2 mL of trypsinzed cell culture were transferred to 15 mL Falcon centrifuge tubes (Invitrogen, Canada) and centrifuged at 200 x g for 5 min to pellet the cells. The supernatants were analyzed for oxidative stress markers (Bahia et al., 2018). A portion of the pelleted cells were resuspended in 350 µL of Buffer RLT containing 1% ?-Mercaptoethanol (Qiagen's RNeasy Mini kit; Qiagen Inc, Mississauga, ON) then frozen at -80oC until processing.  Frozen HLE cells were thawed on ice and mixed well by pipetting. The lysate was transferred directly onto a QIAshredder spin column (Qiagen Inc), placed in a 2 mL collection tube and centrifuged for 2 min at ~12,000 g. A volume of 350 µL of 70% ethanol was added. Total RNA was then extracted using the RNeasy Mini kit according to the manufacturer's instructions (Qiagen Inc), with the addition of Qiagen's On-Column RNase-free DNase (Qiagen Inc) to eliminate any remaining DNA contamination. All total RNA sample concentrations were determined spectrophotometrically using a Nanodrop (Fisher Scientific, Ottawa, ON) (OD ratio of A260:A280). Total RNA (1 µg) was ribodepleted using Ribo-Zero Gold reagent (Illumina, San Diego, CA). Next-Generation Sequencing (NGS) libraries were prepared using Illumina TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA) according to the manufacturer's protocols.