GSM8531293: BD114; Homo sapiens; RNA-Seq
TRIzol method. Homogenized samples were thawed on ice followed by the addition of 200µL of chloroform and thorough mixing, then a 5 min incubation at room temperature, followed by centrifugation for 15 min at 40ºC at 12000g. The clear, aqueous top layer was collected and total RNA was precipitated by adding 100% cold ethanol to the extracted portion until a final concentration of 35% ethanol was achieved. Samples were then incubated for 30 minutes at 20C and centrifuged for 15 minutes at 4C at 16000g. 75% ethanol was used to wash the RNA pellet twice, and the RNA pellet was left to air-dry after which it was resuspended in 20µL of nuclease-free, ultra-pure diethlpyrocarbonate-treated water. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic organisms or eukaryotic organisms' long-non-coding libraries, rRNA is removed using the Ribo-Zero kit that leaves the mRNA. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.