GSM8946663: ICC20; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy Plus Kit (Qiagen). RNA samples were quantified using the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and RNA integrity was assessed with the Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), following the manufacturer's instructions. Briefly, mRNA enrichment was performed using Oligo(dT) beads, followed by fragmentation for 15 minutes at 94°C. First- and second-strand cDNA synthesis was then carried out. The resulting cDNA fragments were end-repaired, adenylated at the 3' ends, and ligated with universal adapters. This was followed by index addition and library enrichment through limited-cycle PCR.The sequencing libraries were validated using the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA) and quantified using the Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA), as well as quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). Libraries were clustered onto flowcell lanes, and after clustering, the flowcell was loaded onto an Illumina instrument, following the manufacturer's protocol. Sequencing was performed in a 2 × 150 bp Paired-End (PE) configuration.