GSM9068003: HCC, CDK18KO, R2; Mus musculus; RNA-Seq
Total RNA was extracted from snap-frozen liver tumor tissue using the QIAGEN RNeasy Mini Kit. Briefly, tumors were mechanically disrupted by chopping in liquid nitrogen, followed by homogenization in RLT buffer (supplemented with β-mercaptoethanol) using a rotor-stator homogenizer. Lysates were processed according to the manufacturer's protocol, including on-column DNase I digestion (QIAGEN RNase-Free DNase). RNA integrity was verified via Agilent Bioanalyzer (RIN ≥ 8.0) Provided total RNA samples (300ng, with average RIN/RQS 8.3, range 7.5-8.8) were converted into sequencing libraries using the "QuantSeq 3' mRNA-Seq V2 Library Prep Kit (FWD) for Illumina" (Lexogen, Cat.No. 191). Briefly, library generation is initiated by reverse transcription with oligodT priming, followed by a random-primed second strand synthesis. A UMI Second Strand Synthesis module was used, in which random primers featuring a 6 nt long Unique Molecular Identifier tag are used {to be addressed for bias correction at data analysis}. Directional cDNA libraries, stranded in the sense orientation {--library-type fr-secondstrand in TopHat, --stranded=yes in HTSeq, Reverse for HISAT}, were completed by PCR and sequenced in a single read format on an Illumina NovaSeq X (with NovaSeq X Series Reagent Kits) and siingle read 151 bases