GSM8968390: RPE-1 DDX17 intronic deletion hgRNA 1, replicate 1; Homo sapiens; RNA-Seq
RNA was extracted using RNeasy Mini Kit (Qiagen #74106). Briefly, cells were washed in PBS twice and lysed directly in the vessel using RLT buffer supplemented with 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. Biological duplicates were processed for RNA-Seq. Stranded/directional total RNA-Seq (ribo-depletion) libraries were prepared as follows. RNA concentration was measured using Qubit RNA BR (cat # Q10211, Thermo Fisher Scientific Inc., Waltham, USA) and RNA quality was assessed from 1 ng input on the Bioanalyzer RNA 6000 Pico kit (cat # 5067-1513, Agilent Technologies Inc., Santa Clara, USA). RIN score ranged from 8.4- 9.8 ; with a median RIN score of 9.6. For library preparation, 300 ng of DNase-treated RNA per sample was first subjected to rRNA depletion (H/M/R) and then processed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat #E7760L; New England Biolabs; protocol 6.0_7/24), with 7.5 minutes of fragmentation at 94 °C and 12 cycles of amplification. To assess final library quality, 1 μl of the library was analyzed on the Agilent Bioanalyzer dsDNA High Sensitivity chip (cat # 5067-4626, Agilent Technologies Inc., Santa Clara, USA). The libraries were quantified using the Qubit 1X dsDNA HS (cat # Q33231, Thermo Fisher Scientific Inc., Waltham, USA) and were pooled at equimolar ratios after adjusting for fragment size. The final pool, with an average library size of ~395 bp was run on the Bioanalyzer and quantified using NEBNext Library Quant Kit for Illumina (cat # E7630L, New England Biolabs, Ipswich, USA).