GSM7794038: BEAS-2B cells, CrudeF, 24hr; Homo sapiens; RNA-Seq
Added 1 ml Tri reagent (Sigma-Aldrich) to the cell culture and incubated for 5 minutes, followed by adding 200μl chloroform, vortexing for 15 seconds, and incubating for 15 minutes. Subsequently, centrifuged under 16,558 xg for 15 minutes and take the supernatant. Added 500μl isopropanol and incubated for 10 minutes, then centrifuged for 30 minutes. Discarded supernatant, added 1 ml 75% ethanol to clean the pellet, and centrifuged again for 30 minutes. Following the discarded supernatant and drying of the pellet, add 25 μl Diethyl pyrocarbonate (DEPC) to restore the pellet. The mRNAs were isolated from total RNAs with poly-T probes and were fragmented before first-strand cDNA synthesis with random primers and reverse transcriptase. The DNA-RNA hybrid products were then synthesized in the second strand cDNAs with DNA polymerase I and ligated adaptors into both ends of cDNAs.