GSM9215846: 1H6-neu-only; Homo sapiens; RNA-Seq
Total RNA (100 ng) was used to prepare sequencing libraries with the RNA HyperPrep Kit with RiboErase (HMR) (Roche Sequencing Solutions), following the manufacturer's protocol. Ribosomal RNA (rRNA) was first depleted, and the remaining RNA was treated with DNase to remove genomic DNA contamination. The RNA was then purified, fragmented, and primed for complementary DNA (cDNA) synthesis.First-strand cDNA synthesis was performed using random primers, followed by second-strand synthesis, during which dUTP was incorporated in place of dTTP to enable strand-specificity. The resulting double-stranded (ds) cDNA was purified using Pure Beads (KAPA Biosystems). To facilitate adapter ligation, an adenine (A) overhang was added to the 3' ends of the blunt fragments, and indexing adapters with a complementary thymine (T) overhang were ligated. Adapter-ligated libraries were amplified by PCR, purified using Pure Beads, and assessed for fragment size distribution using the Agilent 4200 TapeStation D1000 ScreenTape (Agilent Technologies).Library quantification was performed using the KAPA qPCR kit (KAPA Biosystems), and libraries were pooled equimolarly based on the experimental design. The pooled libraries were denatured, diluted to 350 pM, and spiked with 1% PhiX control. Sequencing was conducted on an Illumina NovaSeq 6000 system using 100 bp paired-end reads, following the manufacturer's protocol. On average, 50 million paired-end reads were generated per sample.
GEO
GSM9215846_r1