Qubit rna hs high sensitivity assay kit

Total RNA extraction from 2 independent BJAB-Lck, BJAB-Lyn and -Dox cell cultures was performed using TRIzol (Thermo Fischer Scientific) according to the manufacturer’s protocol and was followed by DNase I (NEB) treatment. The quality of the RNA was visualized using agarose gel electrophoresis. Ribosomal RNA was depleted using the RiboMinusTM Eukaryote Kit v2 (Thermo Fisher Scientific). RNAs was quantified using the Qubit RNA HS (High Sensitivity) Assay Kit (Thermo Fisher Scientific) with the Qubit Fluorometer. cDNA libraries were prepared from 100 ng of rRNA-depleted total RNA using the Ion Total RNA-Seq v2 Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. In brief, RNAs were digested with RNase III and the produced 200nt RNA fragments were successively treated for adapter ligation, reverse transcription, and 14 cycles of PCR amplification using the Ion Xpress™ RNA-Seq Barcode 1-16 Kit (Thermo Fisher Scientific). Yield distribution of the libraries was measured with the Qubit 1x dsDNA HS Assay Kit (Thermo Fisher Scientific) and size distribution was assessed using the Agilent High Sensitivity DNA Kit on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed using an Ion 540™ chip and the 540™ Chef kit on an Ion GeneStudio S5 sequencer.