GSM4498747: U2OS_plus_Aphidicolin_RNA_replicate_1; Homo sapiens; RNA-Seq
Add Trizol directly to the plate to lyse cells.To each Trizol-lysed sample, add 0.2 ml chloroform per1 ml of Trizol used initially and shake vigorously for 5–10 s. Centrifuge at 4°C for 15 min at 12,000g and Transfer the upper aqueous layer to a new EP tube and add 0.5 ml isopropanol per 1 ml of Trizolused initially.Incubate at room temperature for 10 min before centrifuging at 4°C for 10 min at 12,000g to pellet RNA. Remove supernatant and wash pellet by adding 1 ml of 75%ethanol per 1 ml of Trizol used initially. Centrifuge at 4°C for 5 min at 7500g. Remove supernatant and invert tube to allow pellet to dryslightly. Resuspend the pellet in 200ll DEPC-water andincubate at 55°C for 10 min to ensure RNA is fully dissolved. Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular.