title: GSM9160488: new-onset cancer patient 2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using the Quick-DNA/RNA Viral Kit (Zymo Research), following the manufacturer's protocol for whole blood RNA extraction. All procedures were conducted in a biosafety level 2 (BSL-2) cabinet (Esco Labculture A2). Briefly, lysis buffer was added to blood samples to inactivate pathogens, followed by ethanol precipitation and column-based RNA purification. Eluted RNA was quantified and stored at −80°C until library preparation. RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA Kit, incorporating ribosomal RNA depletion and strand-specific cDNA synthesis. RNA was fragmented enzymatically, reverse-transcribed to first-strand cDNA, and converted to double-stranded cDNA. Following end repair and adenylation, Illumina-specific adapters were ligated, and libraries were amplified by PCR. Quality and fragment size distribution were assessed using the High Sensitivity DNA kits on the TapeStation 4200 (Agilent Technologies). Libraries were stored at −20°C prior to sequencing. Normalized and pooled libraries were sequenced on the Illumina NextSeq 550 platform using the NextSeq 500/550 High Output Kit v2.5 (300 cycles), targeting a coverage depth of 200× per sample. PhiX was added at 1% as an internal control. Paired-end sequencing reads were collected, and sequencing quality metrics (e.g., Q30 scores, cluster density) were monitored in real-time using Illumina's Sequencing Analysis Viewer.