GSM9980666: Human nucleus pulposus cells, si-YBX1, biological replicate 3; Homo sapiens; RNA-Seq
Cells were lysed using TRIzol reagent. Total RNA was extracted using a SteadyPure RNA Extraction Kit (AG, AG21024). RNA concentration and purity were assessed spectrophotometrically, RNA integrity was assessed by agarose gel electrophoresis, and RNA integrity values were measured using an Agilent 5300 system. Approximately 1 ug total RNA was used for library preparation. Poly(A)-containing mRNA was enriched from total RNA using oligo(dT) magnetic beads and fragmented to approximately 300 bp. First-strand cDNA was synthesized using random primers, followed by second-strand synthesis with dUTP replacing dTTP. After end repair, A-tailing and adapter ligation, the dUTP-containing second strand was degraded using uracil-DNA glycosylase. The remaining strand was PCR amplified and size selected to generate stranded mRNA-seq libraries. Libraries were sequenced on an Illumina NovaSeq 6000 instrument using paired-end 151-base reads.