GSM9407333: DMSO, IP, rep2; Homo sapiens; OTHER
Treat H69 cells with either DMSO or FB23-2 and lyse them to extract total RNA. Isolate polyA RNA from the total RNA using magnetic Oligo(dT) beads. Elute the purified mRNA from the beads in RNase-free water. Check the concentration and quality of the mRNA using a fluorometer and bioanalyzer. Store the final polyA RNA at -80°C for library preparation and sequencing. Following polyA RNA extraction from DMSO or FB23-2 treated H69 cells, proceed with library construction by first fragmenting the mRNA and synthesizing double-stranded cDNA. Then, perform end repair, dA-tailing, and ligation of indexed sequencing adapters to the cDNA fragments. Amplify the adapter-ligated library via a limited-cycle PCR to enrich for properly constructed fragments. Clean up the final product using a bead-based purification system to select for the correct insert size. Finally, validate the library's concentration and size distribution by fluorometry and bioanalyzer before pooling and sequencing.