GSM9423313: rectus femoris, 1d after operation, patient 5, rep3; Homo sapiens; RNA-Seq
RNA was extracted from frozen tissue samples (~30mg) using the HiPure Viral RNA Kit (Magen, China). RNA concentration was quantified using a fluorometer (Qubit 4.0; Thermo Fisher Scientific). Complementary DNA (cDNA) synthesis was performed using previously isolated mRNA and the Mint kit (Evrogen, Russia). The procedure fol-lowed the manufacturer's protocol, including the addition of IP solution to enhance synthesis efficiency. Subsequently, the synthesized cDNA was amplified using the Encyclo Plus PCR Kit (Evrogen, Russia) with universal primers. To verify amplification success, the resulting products were visualized via horizontal agarose gel electrophoresis. For library preparation, 200 ng of cDNA from each sample was used. Sequencing libraries were constructed from the obtained amplicons using the Ligation Sequencing Kit (SQK LSK109, Oxford Nanopore Technologies, UK), following the manufacturer's pro-tocols. During the DNA end preparation step, End Prep Mix was added to the samples. The mixture was thoroughly mixed by pipetting, and droplets on the tube walls were removed via brief centrifugation. The reaction was carried out in a thermal cycler. After incubation, the mixture was purified using magnetic beads (VAHTS DNA Clean Beads, Vazyme, China). The DNA was eluted in 10 µL of Milli Q water, and its concentration was measured using a Qubit 4 fluorometer (Thermo Fisher Scientific, USA) with the QuDye HS Assay Kit (Lumiprobe, Russia). cDNA libraries were multiplexed for sequencing via native barcoding using the PCR Barcoding Expansion 1–96 kit (EXP PBC096, Oxford Nanopore Technologies, UK), according to the manufacturer's protocol. Samples were purified with VAHTS DNA Clean Beads (Vazyme, China). The concentration of the pooled samples was measured on the Qubit 4. Following ligation, the mixture was cleaned with magnetic beads, washed with SFB (by removing from the magnet and resuspending the beads), and eluted in EB buffer. The final library concentration was verified using the Qubit 4, after which the library was loaded onto the sequencing flow cell.