GSM8968264: HAP1 cycloheximinde treatment, replicate 2; Homo sapiens; RNA-Seq
Total RNA was extracted from HAP1 and RPE-1 cells using the RNeasy Mini Kit (Qiagen #74106). Briefly, cells were washed twice in PBS and lysed directly in the culture vessel using RLT buffer supplemented with the reducing agent β-mercaptoethanol (Milipore, #444203-250ML). All RNA samples were DNase-treated using RNase-Free DNase Set (Qiagen, #79256) according to the manufacturer's protocol. Stranded/directional mRNA-Seq libraries were prepared as follows. DNase-treated total RNA was assessed using Qubit RNA BR (cat # Q10211, Thermo Fisher Scientific Inc., Waltham, USA) and RNA integrity was evaluated using the Bioanalyzer RNA 6000 Pico kit (cat # 5067-1513, Agilent Technologies Inc., Santa Clara, USA) with all samples exhibiting high quality (RIN ≥ 9, median RIN = 9.7). For each sample, 500 ng of total RNA was used to prepare libraries with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat # E7760L; New England Biolabs; protocol 6.0_7/24) incorporating Poly (A) selection, with 7.5 minutes of fragmentation at 94 °C and 11 cycles of amplification. Library quality was verified by running 1 μl of each final library on an Agilent Bioanalyzer using the High Sensitivity dsDNA chip (cat #5067-4626, Agilent Technologies). The libraries were quantified using the Quant-iT dsDNA high-sensitivity (cat # Q33120, Thermo Fisher Scientific Inc., Waltham, USA), then normalized and pooled at equimolar ratios after size-adjustment. The pooled library had an average library size of 502 bp.