quantseq mRNA seq: svct2
Total RNA was isolated using Trizol reagent (Invitrogen). RNA quality was assessed by Agilent TapeStation 4000 system (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using ND-2000 Spectrophotometer (Thermo Inc., DE, USA).For library preparation of control and test samples, the QuantSeq 3 mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) was employed according to the manufacturers protocol. Briefly, total RNA was combined with an oligo-dT primer that included an Illumina-compatible sequence at its 5 end, followed by reverse transcription. After RNA template degradation, second-strand synthesis was initiated with a random primer carrying an Illumina-compatible linker sequence at the 5 end. The double-stranded library was purified using magnetic beads to eliminate residual reaction components. Subsequently, the library was amplified to add the full adapter sequences necessary for cluster generation. The finalized library was further purified to remove PCR contaminants. High-throughput sequencing was performed as single-end 75 sequencing using NextSeq 550 (Illumina, Inc., USA).