GSM9126947: V-Br-24; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA; cat. no. 74104), according to the manufacturer's instructions. RNA concentration and purity were initially measured using the NanoDrop 8000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Sequencing libraries were constructed using the NEBNext Ultra II RNA Library Preparation Kit for Illumina (New England Biolabs, Ipswich, MA, USA), as per the manufacturer's recommendations. Briefly, RNA samples were fragmented at 94 °C for 15 minutes, followed by first and second-strand cDNA synthesis using primers containing random priming sites. Subsequent steps included end repair and adenylation of cDNA fragments at the 3' ends, adapter ligation, indexing, and enrichment via limited-cycle PCR. Final sequencing libraries were validated using the Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA) and quantified with the Qubit 3.0 Fluorometer and quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). Prepared libraries were clustered onto a lane of a NovaSeq flowcell, which was subsequently loaded onto an Illumina NovaSeq sequencing instrument following the manufacturer's instructions. Sequencing was performed using a paired-end (PE) configuration of 2×150 bp reads. Image analysis and base calling were executed by the instrument's control software. The raw sequencing output in .bcl file format was converted to FASTQ files and demultiplexed using Illumina's bcl2fastq software version 2.17, allowing one mismatch during index sequence identification.