GSM9336348: CBOS1_DMSO_3; Homo sapiens; RNA-Seq
After treatment, a minimum of 24 spheroids were collected for each condition and transferred into Eppendorf tubes. They were rinsed twice with 1× PBS (Gibco, 14190-144) and then centrifuged at 500 g for 4 minutes. RNA extraction was performed by lysing the spheroids using the PureLink RNA Mini Kit (Invitrogen, 12183018A), following the manufacturer's protocol, along with on-column DNase treatment using the PureLink DNase kit (Invitrogen, 12185010). The concentration and purity of the isolated RNA were assessed based on absorbance readings at 260/280 nm using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies). Total RNA samples were submitted to NovoGene for Human mRNA Sequencing. Library preparation and sequencing were carried out by NovoGene using their standard mRNA-seq workflow. Briefly, polyadenylated mRNA was enriched from total RNA using oligo(dT)-attached magnetic beads and then fragmented. First-strand cDNA synthesis was performed using random hexamer primers, followed by second-strand synthesis to generate double-stranded cDNA. Libraries were constructed through end repair, A-tailing, adapter ligation, PCR amplification, and purification. Final libraries underwent quality assessment at NovoGene prior to sequencing by Qubit and real-time PCR for quantification and bioanalyzer size distribution detection. Quantified libraries were pooled and Sequencing was performed by NovoGene on an Illumina NovaSeq X Plus platform using paired-end 150 bp (PE150) reads.