GSM9149590: MDCK cells, Mel56 (10 µM), N=3; Canis lupus; RNA-Seq
Total RNA was extracted from cell lysates using an RNeasy Mini Kit (Qiagen, GmbH, Germany). Total RNA concentration was calculated using Quant-IT RiboGreen (Thermo Fisher Scientific). To assess the integrity of total RNA, samples were run on a TapeStation RNA Screentape (Agilent Technologies, CA, USA). Only high-quality RNA preparations (RIN greater than 7.0) were used for RNA library construction. A library was independently prepared with 1 µg of total RNA for each sample using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, CA, USA). The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T-attached magnetic beads. Following purification, mRNA was fragmented into small pieces using divalent cations at elevated temperatures. Cleaved RNA fragments were copied into first-strand cDNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) and random primers. This was followed by the synthesis of second-strand cDNA using DNA Polymerase I, RNase H, and dUTP. These cDNA fragments underwent an end repair process, followed by the addition of a single 'A' base and ligation of the adapters. The products were purified and enriched using PCR to create a final cDNA library. Libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS, MA, USA) and qualified using a TapeStation D1000 ScreenTape (Agilent Technologies).